Novel compounds

ABSTRACT

The present invention relates to an antibody which has multiple specificities. In particular the antibody of the present invention binds to (cross react with) human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional application60/912,229 filed Apr. 17, 2007 and 61/044,132 filed Apr. 11, 2008, whichare incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to an antibody which has multiplespecificities. In particular the antibody of the present invention bindsto (cross react with) human IL-8, Gro-alpha, Gro-beta, Gro-gamma, andENA-78. The present invention also concerns with methods of treatingdiseases or disorders characterised by elevated or unbalanced level ofone or more of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78,particularly COPD, osteoarthritis, rheumatoid arthritis, erosivearthritis, asthma, atherosclerosis, inflammatory bowel disease,psoriasis, transplant rejection, gout, cancer, acute lung injury, acutelung disease, sepsis, ARDS, peripheral artery disease, systemicsclerosis, neonatal respiratory distress syndrome, exacerbation ofasthma and COPD, cystic fibrosis, diffuse panbronchiolitis, reperfusioninjury, and/or endometriosis with said antibody.

BACKGROUND OF THE INVENTION

Published data and reports indicate that the members of the ELRCXCsubfamily of CXCL chemokines are elevated in a number of diseases. Thereare a total of 16 CXCL family members. The chemokines are reported to beup-regulated in a number of inflammatory diseases, including COPD, inwhich CXCL1-3, 5, and 8, also known as Gro-1-α, -β, -γ (Haskill, S., etal. Proc. Natl. Acad. Sci., 1990: 87, 7732-7736), ENA-78 (Wang, D. andRichmond, A., Cytokine Reference. Oppenheim, J. J. and Feldman, M. ed.,Academic Press, London, 1023-1027, Powerm C. A. et al. Gene., 1994: 151,333-334), and IL-8 (Iizasa, H. and Matsushima, K., Cytokine Reference.Oppenheim, J. J. and Feldman, M. ed., Academic Press, London, 1061-1067,Matsushima, K. et al., J. Exp. Med. 1988: 167, 1883-1893) respectively(Am. J. Respir. Crit Care Med., 163: 349-355, 2001, Am. J. Respir. CritCare Med., 168: 968-975, 2003, Thorax, 57: 590-595, 2002). It has bepostulated that prolonged and elevated expression of these chemokinescould be involved in the development of diseases such as COPD,osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma,atherosclerosis, inflammatory bowel disease, psoriasis, transplantrejection, gout, cancer, acute lung injury, acute lung disease, sepsis,ARDS, peripheral artery disease, systemic sclerosis, neonatalrespiratory distress syndrome, exacerbation of asthma and COPD, cysticfibrosis, diffuse panbronchiolitis, reperfusion injury, orendometriosis. These CXC chemokines are known to stimulate neutrophilchemotaxis by engaging and activating the CXCR1 and/or CXCR2 receptors.Thus the inhibition of these chemokines could prevent inflammatory cellsfrom infiltrating the lung tissue and thus prevent tissue damage. Thepresent invention is directed to inhibiting the activation of CXCR1 andCXCR2 receptors by using an antibody having the ability to bind to humanIL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78, i.e. a penta-specificantibody.

SUMMARY OF THE INVENTION

The present invention relates to an antibody (immunoglobulin) which hasmultiple specificities contained within one immunoglobulin. Inparticular the antibody of the present invention binds to (cross reactwith) human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78. Thepresent invention also concerns with methods of treating diseases ordisorders characterised by elevated or unbalanced level of one or moreof human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78, particularlyCOPD, osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma,atherosclerosis, inflammatory bowel disease, psoriasis, transplantrejection, gout, cancer, acute lung injury, acute lung disease, sepsis,ARDS, peripheral artery disease, systemic sclerosis, neonatalrespiratory distress syndrome, exacerbation of asthma and COPD, cysticfibrosis, diffuse panbronchiolitis, reperfusion injury, and/orendometriosis with said antibody.

In one aspect, the present invention relates to an isolated antibodywhich has multiple specificity to (or cross reacts with) human IL-8,Gro-alpha, Gro-beta, Gro-gamma, and ENA-78; thus we define the antibodyof the present invention as penta-specific (or pan-ELR) antibody. Thedefinition of antibody includes an antigen binding portion (or fragment)of the antibody such that the antigen binding portion (or fragment)binds to human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78. Thepenta-specific antibody of the invention is preferably murinemonoclonal, chimeric, human or humanized. For avoidance of doubt, thepenta-specific antibody of the present invention need not bind solely tohuman IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78 antigens, but itmay also to bind to other related proteins (such as human GCP-2, or mayeven bind to non-human orthologues of IL-8, Gro-alpha, Gro-beta,Gro-gamma, ENA-78, and GCP-2); in other words, the penta-specificantibody of the present invention minimally binds to human IL-8,Gro-alpha, Gro-beta, Gro-gamma, and ENA-78.

Preferably, a penta-specific antibody of the present invention binds toeach of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78 withequilibrium constant, KD, values of less than 10⁻⁷ M, more preferablyless than 10⁻⁸ M, and even more preferably less than 10⁻⁹ M asdetermined by surface plasmon resonance. Typically surface plasmonresonance measurement is conducted as described below.

In one embodiment the present invention comprises a method of decreasingthe neutrophil chemotaxis through inhibition of CXCR1 and CXCR2 receptoractivation by neutralizing human IL-8, Gro-alpha, Gro-beta, Gro-gamma,GCP-2 and ENA-78 with a penta-specific antibody of the presentinvention.

In one embodiment the present invention relates to a method ofdecreasing the neutrophil chemotaxis in a patient in need thereof byadministering a penta-specific antibody of the present invention.

In one embodiment, a penta-specific antibody binds within epitope ofKELRCQCIKTYSKP (SEQ ID NO: 54) in human IL-8.

In one embodiment, the penta-specific antibody of the present inventionis generated by a method comprising the steps of using RIMMs(Kilpatrick, K. E., et al. Hybridoma. 1997: 16, 381) type protocol usinga mixture (cocktail) of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, andENA-78 together with a set of five multiple antigenic peptides (MAPs)each MAP unit having one separate sequence from polypeptides of ID NOs:49-53.

LATELRSQSLQTLQG SEQ ID NO: 49 SAKELRSQSIKTYSK SEQ ID NO: 50LRELRSVSLQTTQG SEQ ID NO: 51 SPGPHSAQTEVIAT SEQ ID NO: 52 ESGPHSANTEIIVKSEQ ID NO: 53

Without being bound by theory, MAPs serve two functions within theimmunization protocol. First, MAPs allow for a selective multiplepresentation of a known target amino acid sequence to the host immunesystem. Secondly, the increase in mass, due to multiple copies of thesequence linked via a core, such as, but not limited to lysine,increases the immunogenicity of the sequence over that of individualpeptides (Francis, J. P., et al., Immunology, 1991: 73; 249, Schott, M.E., et al., Cell. Immuno. 1996: 174: 199-209, Tam, J. P. Proc. Natl.Acad. Sci. 1988: 85; 5409-5413).

The MAPs used to generate this invention are comprised of multiplecopies of the conserved target sequences (e.g. SEQ ID NOs: 49-53) foundwith and around the ELRCXC and GPHCA regions of target chemokines.Exemplary MAP set is depicted in FIG. 1.

In one embodiment, a penta-specific antibody of the present invention isgenerated by a method comprising the steps of:

-   -   a. injecting into a mouse a mixture of human IL-8, Gro-alpha,        Gro-beta, Gro-gamma, and ENA-78 in complete Freund's adjuvant        (cFA);    -   b. injecting into the mouse a mixture of human IL-8, Gro-alpha,        Gro-beta, Gro-gamma, and ENA-78 in incomplete Freund's adjuvant        (iFA); and    -   c. injecting into the mouse a mixture of human IL-8, Gro-alpha,        Gro-beta, Gro-gamma, and ENA-78, and a set of five multiple        antigenic peptides (MAPs), each MAP unit having one separate        sequence from polypeptides of ID NOs: 49-53 in incomplete        Freund's adjuvant;    -   d. isolating B cells from the mouse;    -   e. fusing the B cells with myeloma cells to form immortal        hybridoma cells that secrete the desired penta-specific        antibody; and    -   f. isolating the penta-specific antibody from the culture        supernatant of the hybridoma.    -   If desired, one can optionally inject into the mouse a mixture        of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78, and a        set of MAPs comprising amino acid sequences of SEQ ID NOs: 49-53        in PBS between steps c and d.

In another embodiment, a penta-specific antibody of the presentinvention is generated by a method comprising the steps of:

-   -   a. injecting into a mouse a set of five multiple antigenic        peptides (MAPs) each MAP unit having one separate sequence from        polypeptides of ID NOs: 49-53 (hereinafter also referred to as        the MAP set) in complete Freund's adjuvant;    -   b. injecting into the mouse the MAP set in incomplete Freund        adjuvant;    -   c. injecting into the mouse a mixture of all human IL-8,        Gro-alpha, Gro-beta, Gro-gamma, and ENA-78, and the MAP set in        incomplete Freund's adjuvant;    -   d. isolating B cells from the mouse; and    -   e. fusing the B cells with myeloma cells to form immortal        hybridoma cells that secrete the desired penta-specific        antibody; and    -   f. isolating the penta-specific antibody from the culture        supernatant of the hybridoma.    -   If desired, one can optionally inject into the mouse a mixture        of human IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78, and a        set of MAPs having SEQ ID NOs: 49-53 in PBS between steps c and        d.

In another embodiment, the present invention concerns a penta-specificantibody made by the foregoing process.

In one embodiment, a penta-specific antibody has heavy and light chainvariable regions encoded by nucleotide sequences comprising sequences ofSEQ ID NO: 1 and SEQ ID NO:3, respectively, or conservative sequencemodifications thereof.

In one embodiment, a penta-specific antibody has heavy and light chainvariable regions encoded by nucleotide sequences comprising sequences ofSEQ ID NO:5 and SEQ ID NO:7, respectively, or conservative sequencemodifications thereof.

In one embodiment, a penta-specific antibody has heavy chain variableregion encoded by a nucleotide sequence comprising sequence of SEQ IDNO:9, or conservative sequence modifications thereof.

In one embodiment, a penta-specific antibody has heavy and light chainvariable regions comprising the amino acid sequences of SEQ ID NO:2 andSEQ ID NO:4, respectively, or conservative sequence modificationsthereof.

In one embodiment, a penta-specific antibody has heavy and light chainvariable regions comprising polypeptides which are at least 90%, 95%,98% or 99% identical to the amino acid sequences of SEQ ID NO:2 and SEQID NO:4, respectively.

In one embodiment, a penta-specific antibody has heavy and light chainvariable regions comprising the amino acid sequences of SEQ ID NO:6 andSEQ ID NO:8, respectively, or conservative sequence modificationsthereof.

In one embodiment, a penta-specific antibody has heavy and light chainvariable regions comprising polypeptides which are at least 90%, 95%,98% or 99% identical to the amino acid sequences of SEQ ID NO:6 and SEQID NO:8, respectively.

In one embodiment, a penta-specific antibody has heavy and lightvariable regions comprising the amino acid sequences of SEQ ID NOs:10and 12, respectively, or conservative sequence modifications thereof.

In one embodiment, a penta-specific antibody has heavy and light chainvariable regions comprising polypeptide sequences which are at least90%, 95%, 98% or 99% identical to the amino acid sequences of SEQ IDNOs:10 and 12, respectively.

In one embodiment, a penta-specific antibody comprises at least onevariable region selected from (i) the amino acid SEQ ID NO: 2, 4, 6, 8,10, or 12; or (ii) an amino acid sequence which is at least 90%, 95%,98% or 99% identical to any one of the amino acid sequences of (i)above.

In one embodiment, a penta-specific antibody comprises CDR sequences ofSEQ ID NOs: 13, 14, 15, 16, 17, and 18; or one or more of the CDRsequences can be conservative sequence modifications of the sequencesSEQ ID NOs: 13, 14, 15, 16, 17, and 18.

In one embodiment, the present invention relates to hybridoma ortransfectoma which produces a penta-specific antibody which comprisesCDR sequences of SEQ ID NOs: 13, 14, 15, 16, 17, and 18.In one embodiment, the present invention relates to a recombinanteukaryotic or prokaryotic cell which produces a penta-specific antibodywhich comprises CDR sequences of SEQ ID NOs: 13, 14, 15, 16, 17, and 18.

In one embodiment, a penta-specific antibody comprises at least one CDRsequence selected from (i) SEQ ID NO: 13, 14, 15, 16, 17, or 18; or (ii)a conservative sequence modification of the sequences listed in (i).

In one embodiment, a penta-specific antibody comprises a polypeptide ofSEQ ID NO:15.

In one embodiment, a penta-specific antibody comprises at least four CDRsequences selected from the group consisting of SEQ ID NOs: 13, 14, 15,16, 17, and 18; or one or more of the CDR sequences can be conservativesequence modifications of the sequences listed in SEQ ID NOs: 13, 14,15, 16, 17, and 18.

In one embodiment, a penta-specific antibody comprises heavy and lightchain variable regions which comprise the CDR amino acid sequences ofSEQ ID NOs: 13, 14, and 15, and SEQ ID NOs: 16, 17, and 18,respectively.

In one embodiment, a penta-specific antibody comprises CDR sequences ofSEQ ID NOs: 19, 20, 21, 22, 23, and 24; or one or more the CDR sequencescan be conservative sequence modifications of the sequences listed inSEQ ID NOs: 19, 20, 21, 22, 23, and 24.

In one embodiment, the present invention relates to an hybridoma ortransfectoma which produces a penta-specific antibody which comprisesCDR sequences of SEQ ID NOs: 19, 20, 21, 22, 23, and 24.

In one embodiment, the present invention relates to a recombinanteukaryotic or a prokaryotic cell which produces a penta-specificantibody which comprises CDR sequences of SEQ ID NOs: 19, 20, 21, 22,23, and 24.

In one embodiment, a penta-specific antibody comprises at least one CDRsequence selected from (i) SEQ ID NO: 19, 20, 21, 22, 23, or 24; or (ii)a conservative sequence modification of the sequences listed in (i).

In one embodiment, a penta-specific antibody comprises a polypeptide ofSEQ ID NO:21.

In one embodiment, a penta-specific antibody comprises at least four CDRsequences selected from the group consisting of: SEQ ID NOs: 19, 20, 21,22, 23, and 24; or one or more of the CDR sequences can be conservativesequence modifications of the sequences listed in SEQ ID NOs: 19, 20,21, 22, 23, and 24.

In one embodiment, a penta-specific antibody comprises heavy and lightchain variable regions which comprise the CDR amino acid sequences ofSEQ ID NOs: 19, 20, and 21, and SEQ ID NOs: 22, 23, and 24,respectively.

In one embodiment, a penta-specific antibody comprises CDR sequences ofSEQ ID NOs: 25, 26, 27, 28, 29, and 30; or one or more the CDR sequencescan be conservative sequence modifications of the sequences listed inSEQ ID NOs: 25, 26, 27, 28, 29, and 30.

In one embodiment, the present invention relates to an hybridoma ortransfectoma which produces a penta-specific antibody which comprisesCDR sequences of SEQ ID NOs: 25, 26, 27, 28, 29, and 30.

In one embodiment, the present invention relates to a recombinanteukaryotic or prokaryotic cell which produces a penta-specific antibodywhich comprises CDR sequences of SEQ ID NOs: 25, 26, 27, 28, 29 and 30.

In one embodiment, a penta-specific antibody comprises at least one CDRsequence selected from (i) SEQ ID NO: 25, 26, 27, 28, 29, or 30; or (ii)a conservative sequence modification of the sequences listed in (i).

In one embodiment, a penta-specific antibody comprises a polypeptide ofSEQ ID NO:27.

In one embodiment, a penta-specific antibody comprises at least four CDRsequences selected from the group consisting of: SEQ ID NOs: 25, 26, 27,28, 29, and 30; or one or more of the CDR sequences can be conservativemodifications of the sequences listed in SEQ ID NOs: 25, 26, 27, 28, 29,and 30.

In one embodiment, a penta-specific antibody comprises heavy and lightvariable chain regions which comprise the CDR amino acid sequences ofSEQ ID NOs: 25, 26, and 27, and SEQ ID NOs: 28, 29 and 30, respectively.

In one embodiment, the present invention concerns a hybridoma whichproduces a monoclonal antibody having heavy or light chain variableregion encoded by nucleotide sequences comprising nucleotide sequencesof SEQ ID NO:1, 5, or 9, or SEQ ID NO:3 or 7, respectively.

In one embodiment, the present invention concerns a hybridoma whichproduces a monoclonal antibody having heavy or light chain variableregion comprising the amino acid sequences of SEQ ID NO:2, 6, or 10, orSEQ ID NO: 4, 8, or 12, respectively, and conservative sequencemodifications thereof.

In one embodiment, the present invention relates to a recombinanteukaryotic or a prokaryotic host cell which produces a penta-specificantibody having heavy or light variable region which comprise the aminoacid sequences of SEQ ID NO:2, 6, or 10, or SEQ ID NO:4, 8, or 12,respectively, and conservative sequence modifications thereof.

In one embodiment, the present invention relates to an expression vectorcomprising nucleotide sequences encoding a variable heavy or light chainof a penta-specific antibody comprising the CDR sequences of SEQ ID NOs:13, 14, and 15; or SEQ ID NOs: 16, 17, and 18, respectively.

In one embodiment, the present invention relates to an expression vectorcomprising a nucleotide sequence encoding a CDR sequence of apenta-specific antibody selected from SEQ ID NO: 13, 14, 15, 16, 17, or18.

In one embodiment, the present invention relates to an expression vectorcomprising nucleotide sequences encoding at least four CDR sequences ofa penta-specific antibody selected from the group consisting of SEQ IDNOs: 13, 14, 15, 16, 17, and 18.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences of SEQ ID NOs: 31, 32, or 33.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences of SEQ ID NOs: 34, 35, and 36.

In one embodiment the present invention relates to an expression vectorcomprising at least four polynucleotide sequences selected from thegroup of SEQ ID NOs: 31, 32, 33, 34, 35, and 36.

In one embodiment, the present invention relates to an expression vectorcomprising nucleotide sequences encoding a variable heavy or light chainof a penta-specific antibody comprising the CDR sequences of SEQ ID NOs:19, 20, and 21; or 22, 23, and 24, respectively.

In one embodiment, the present invention relates to an expression vectorcomprising a nucleotide sequence encoding a CDR sequence of apenta-specific antibody selected from SEQ ID NO: 19, 20, 21, 22, 23, or24.

In one embodiment, the present invention relates to an expression vectorcomprising nucleotide sequences encoding at least four CDR sequences ofa penta-specific antibody selected from the group consisting of SEQ IDNOs: 19, 20, 21, 22, 23, and 24.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences of SEQ ID NOs: 37, 38, and 39.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences of SEQ ID NOs: 40, 41, and 42.

In one embodiment the present invention relates to an expression vectorcomprising at least four polynucleotide sequences selected from thegroup of SEQ ID NOs: 37, 38, 39, 40, 41, and 42.

In one embodiment, the present invention relates to an expression vectorcomprising nucleotide sequences encoding a variable heavy chain of apenta-specific antibody comprising the CDR sequences of SEQ ID NOs: 25,26, and 27.

In one embodiment, the present invention relates to an expression vectorcomprising a nucleotide sequence encoding a CDR sequence of apenta-specific antibody selected from SEQ ID NO: 25, 26, or 27.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences of SEQ ID NOs: 43, 44, and 45.

In one embodiment, a penta-specific antibody has heavy and light chainsencoded by nucleotide sequences comprising sequences of SEQ ID NO:11,and SEQ ID NO:47, 59, 61, or 63, respectively.

In one embodiment, a penta-specific antibody has heavy and light chainsencoded by nucleotide sequences comprising sequences which are at least90%, 95%, 98% or 99% identical to sequences of SEQ ID NO:11, and SEQ IDNO:47, 59, 61, or 63, respectively.

In one embodiment, a penta-specific antibody has heavy and light chainscomprising the amino acid sequences of SEQ ID NO:46, and SEQ ID NO:48,60, 62, or 64, respectively.

In one embodiment, a penta-specific antibody has heavy and light chainscomprising the amino acid sequences of SEQ ID NO:46 and SEQ ID NO:48,respectively.

In one embodiment, a penta-specific antibody has heavy and light chainscomprising the amino acid sequences of SEQ ID NO:46 and SEQ ID NO:60,respectively.

In one embodiment, a penta-specific antibody has heavy and light chainscomprising the amino acid sequences of SEQ ID NO:46 and SEQ ID NO: 62,respectively.

In one embodiment, a penta-specific antibody has heavy and light chainscomprising the amino acid sequences of SEQ ID NO:46 and SEQ ID NO:64,respectively.

In one embodiment, a penta-specific antibody has heavy and light chainscomprising polypeptides which are at least 90%, 95%, 98% or 99%identical to the amino acid sequences of SEQ ID NO:46, and SEQ ID NO:48,60, 62, or 64, respectively.

In one embodiment, the present invention relates to a recombinanteukaryotic or a prokaryotic host cell which produces a penta-specificantibody having heavy or light chain comprising the amino acid sequenceof SEQ ID NO: 46, or SEQ ID NO:48, 60, 62, or 64, respectively.

In one embodiment, the present invention relates to a recombinanteukaryotic or a prokaryotic host cell which produces a penta-specificantibody having heavy and light chains comprising the amino acidsequences of SEQ ID NO: 46, and SEQ ID NO:48, 60, 62, or 64,respectively.

In one embodiment, the present invention relates to a recombinanteukaryotic or a prokaryotic host cell which produces a penta-specificantibody having heavy or light chain comprising the amino acid sequencewhich is at least 90%, 95%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 46, or SEQ ID NO:48, 60, 62, or 64, respectively.

In one embodiment, the present invention relates to a recombinanteukaryotic or a prokaryotic host cell which produces a penta-specificantibody having heavy and light chains comprising the amino acidsequences which are at least 90%, 95%, 98% or 99% identical to the aminoacid sequences of SEQ ID NO: 46, and SEQ ID NO:48, 60, 62, or 64,respectively.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences comprising sequences of SEQ IDNO:11, and SEQ ID NO:47, 59, 61, or 63, respectively.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences comprising sequences which are atleast 90%, 95%, 98% or 99% identical to sequences of SEQ ID NO:11, andSEQ ID NO:47, 59, 61, or 63, respectively.

In one embodiment, the present invention relates to an expression vectorcomprising a polynucleotide sequence comprising a sequence of SEQ IDNO:11, 47, 59, 61, or 63.

In one embodiment, the present invention relates to an expression vectorcomprising a polynucleotide sequence comprising a sequence which is atleast 90%, 95%, 98% or 99% identical to a sequence of SEQ ID NO:11, 47,59, 61, or 63, respectively.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences which encode a penta-specificantibody comprising heavy and light chains comprising the amino acidsequences of SEQ ID NO:46, and SEQ ID NO:48, 60, 62, or 64,respectively.

In one embodiment, the present invention relates to an expression vectorcomprising polynucleotide sequences which encode a penta-specificantibody comprising heavy and light chains comprising the amino acidsequences which are at least 90%, 95%, 98% or 99% identical to the aminoacid sequences of SEQ ID NO:46, and SEQ ID NO:48, 60, 62, or 64,respectively.

In one embodiment, the present invention relates to an expression vectorcomprising a polynucleotide sequence which encode a polypeptidecomprising the amino acid sequence of SEQ ID NO:46, 48, 60, 62, or 64.

In one embodiment, the present invention relates to an expression vectorcomprising a polynucleotide sequence which encode a polypeptidecomprising an amino acid sequence which is at least 90%, 95%, 98% or 99%identical to the amino acid sequences of SEQ ID NO:46, 48, 60, 62, or64.

In one embodiment the present invention relates to a process forproducing a penta-specific antibody (immunoglobulin) in a single hostcell, comprising the steps of:

-   -   (i) transforming said single host cell with a first DNA sequence        encoding a heavy chain comprising polypeptide of SEQ ID NO: 46;        and a second DNA sequence encoding a light chain comprising a        polypeptide of SEQ ID NO: 48, 60, 62, or 64; and    -   (ii) expressing said first DNA sequence and said second DNA        sequence so that said immunoglobulin heavy and light chains are        produced as separate molecules in said transformed single host        cell;    -   furthermore, this process can be carried out such that said        first and second DNA sequences are present in different vectors        or said first and second DNA sequences are present in a single        vector.

In one embodiment the present invention relates to a process forproducing a penta-specific antibody (immunoglobulin) in a single hostcell, comprising the steps of:

-   -   (i) transforming said single host cell with a first DNA sequence        encoding at least the variable domain of the immunoglobulin        heavy chain comprising CDR domains of SEQ ID NOs: 13, 14, and        15; and a second DNA sequence encoding at least the variable        domain of the immunoglobulin light chain comprising CDR domains        of SEQ ID NOs: 16, 17, and 18; and    -   (ii) expressing said first DNA sequence and said second DNA        sequence so that said immunoglobulin heavy and light chains are        produced as separate molecules in said transformed single host        cell;        furthermore, this process can be carried out such that said        first and second DNA sequences are present in different vectors        or said first and second DNA sequences are present in a single        vector.

In one embodiment the present invention relates to a process forproducing a penta-specific antibody (immunoglobulin) in a single hostcell, comprising the steps of:

-   -   (i) transforming said single host cell with a first DNA sequence        encoding at least the variable domain of the immunoglobulin        heavy chain comprising CDR domains of SEQ ID NOs: 19, 20, and        21; and a second DNA sequence encoding at least the variable        domain of the immunoglobulin light chain comprising CDR domains        of SEQ ID NOs: 22, 23, and 24; and    -   (ii) expressing said first DNA sequence and said second DNA        sequence so that said immunoglobulin heavy and light chains are        produced as separate molecules in said transformed single host        cell;        this process can be carried out such that said first and second        DNA sequences are present in different vectors or said first and        second DNA sequences are present in a single vector.

In one embodiment the present invention relates to an antibody thatfully or partially blocks the binding of any one of the aforementionedpenta-specific antibody to human IL-8, Gro-alpha, Gro-beta, Gro-gamma,ENA-78, and GCP-2 in an immunoassay, such as ELISA assay. In oneembodiment, partial blocking occurs when the antibody blocks the bindingof the penta-specific antibody by more than 10%, 20%, 40% or 50%.

In one embodiment the present invention relates to an antibody thatcompetes with the binding of any of the aforementioned penta-specificantibody to human IL-8, Gro-alpha, Gro-beta, Gro-gamma, ENA-78, andGCP-2.

In one embodiment the present invention relates to an antibody thatfully or partially blocks the binding of any one of the aforementionedpenta-specific antibody to epitope of KELRCQCIKTYSKP (SEQ ID NO: 54) inhuman IL-8 in an immunoassay, such as ELISA assay. In one embodiment,partial blocking occurs when the antibody blocks the binding of thepenta-specific antibody by more than 10%, 20%, 40% or 50%.

In one embodiment the present invention relates to an antibody thatcompetes with the binding of any one of the aforementionedpenta-specific antibody to epitope of KELRCQCIKTYSKP (SEQ ID NO: 54) inhuman IL-8.

In one embodiment, the present invention relates to a compositioncomprising an aforementioned penta-specific antibody and apharmaceutically acceptable carrier.

In one embodiment, the present invention relates to a method of treatingor preventing in a mammal COPD, osteoarthritis, rheumatoid arthritis,erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease,psoriasis, transplant rejection, gout, cancer, acute lung injury, acutelung disease, sepsis, ARDS, peripheral artery disease, systemicsclerosis, neonatal respiratory distress syndrome, exacerbation ofasthma and COPD, cystic fibrosis, diffuse panbronchiolitis, reperfusioninjury, and/or endometriosis comprising administering an effectiveamount of an aforementioned penta-specific antibody to said mammal.

In one embodiment the present invention relates to an aforementionedpenta-specific antibody for use in the treatment of diseases ordisorders characterised by elevated or unbalanced level of one or moreof human IL-8, Gro-alpha, Gro-beta, Gro-gamma, GCP-2 and ENA-78,particularly COPD, osteoarthritis, rheumatoid arthritis, erosivearthritis, asthma, atherosclerosis, inflammatory bowel disease,psoriasis, transplant rejection, gout, cancer, acute lung injury, acutelung disease, sepsis, ARDS, peripheral artery disease, systemicsclerosis, neonatal respiratory distress syndrome, exacerbation ofasthma and COPD, cystic fibrosis, diffuse panbronchiolitis, reperfusioninjury, or endometriosis.

In one aspect, the present invention relates to an aforementionedpenta-specific antibody for use in preventing and/or treating COPD,osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma,atherosclerosis, inflammatory bowel disease, psoriasis, transplantrejection, gout, cancer, acute lung injury, acute lung disease, sepsis,ARDS, peripheral artery disease, systemic sclerosis, neonatalrespiratory distress syndrome, exacerbation of asthma and COPD, cysticfibrosis, diffuse panbronchiolitis, reperfusion injury, and/orendometriosis in a mammal.

In one aspect, the present invention relates to use of an aforementionedpenta-specific antibody in the manufacture of a medicament for use inpreventing and/or treating COPD, osteoarthritis, rheumatoid arthritis,erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease,psoriasis, transplant rejection, gout, cancer, acute lung injury, acutelung disease, sepsis, ARDS, peripheral artery disease, systemicsclerosis, neonatal respiratory distress syndrome, exacerbation ofasthma and COPD, cystic fibrosis, diffuse panbronchiolitis, reperfusioninjury, and/or endometriosis in a mammal.

In one aspect, the present invention relates to use of an aforementionedpenta-specific antibody in the manufacture of a medicament forpreventing and/or treating COPD, osteoarthritis, rheumatoid arthritis,erosive arthritis, asthma, atherosclerosis, inflammatory bowel disease,psoriasis, transplant rejection, gout, cancer, acute lung injury, acutelung disease, sepsis, ARDS, peripheral artery disease, systemicsclerosis, neonatal respiratory distress syndrome, exacerbation ofasthma and COPD, cystic fibrosis, diffuse panbronchiolitis, reperfusioninjury, and/or endometriosis in a mammal.

In one embodiment, above mammal is human.

DESCRIPTION OF FIGURES

FIG. 1 depicts an exemplary set of MAPs to generate a penta-specificantibody. For avoidance of doubt, five MAP peptide units are depicted.Each unit contains one identical amino acid sequence selected fromlinear peptides of SEQ ID NO: 49-53.

FIG. 2 shows flow cytometric data comparing neutrophils present in BALsamples. Sessions 1, 2 and 3 are presented. The black solid bars showthe baselines 5 day pre-LPS challenge. The grey solid bars depict theBAL data 6-hours post LPS challenge. The Chimera Antibody (HcLc)treatment significantly and dose dependently inhibited neutrophilinfiltration. The grey hatched bars represent 24-hour data. (n=6,*p≦0.05, †p≦0.01, error bars represent standard deviation).

FIG. 3 shows hematological analysis of total circulating neutrophils(cell count). IV injection of 1 (open squares, □) and 10 mg/kg (closedcircles, ) of the Chimera Antibody (HcLc) prevented inhaled LPSstimulation of circulating neutrophils. Both 1 and 10 mg/kg treatmentsare significantly reduced compared to the vehicle NHP (†) and 10 mg/kgtreatment was also significantly reduced compared to the 1 mg/kg dose(*). Data are represented as total count per ul of blood (n=6, *p≦0.05vs. 1 mg/kg, †p≦0.01 vs. vehicle, error bars represent standarddeviation).

FIG. 4 shows inhibition human IL-8 stimulated neutrophil activation(increased CD11b surface expression). Various concentrations of thehumanized penta-specific antibodies were pre-incubated with 10 nM hIL-8prior to addition to purified human neutrophils. Data are expressed asmean values of 4 different donors (n=4). Bars represent standard error.All samples are compared to purified neutrophils stimulated with hIL-8only (no mAb prior to addition to purified neutrophils). Humanizedconstruct H0L10 and H0M0 are more effective at inhibiting hIL-8stimulated CD11b surface expression.

FIG. 5A shows mouse 656.35 mAb binding to target human chemokines.

FIG. 5B shows the Chimera Antibody (HcLc) mAb binding to human targetchemokines.

FIG. 5C shows humanized mAbs binding to human target chemokines.

DETAILED DESCRIPTION OF THE INVENTION

An “isolated penta-specific antibody” or simply “penta-specificantibody”, as used herein, is intended to refer to an antibody thatbinds to and therefore cross reacts with human IL-8, Gro-alpha,Gro-beta, Gro-gamma, and ENA-78, and is substantially free of otherantibodies having different antigenic specificities, and furthermore, isa single composition of matter. For avoidance of doubt, thepenta-specific antibody of the present invention need not bind solely tohuman IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78 antigens, but itmay also happen to bind to other related proteins, such as human GCP-2,or may even bind to non-human orthologues of IL-8, Gro-alpha, Gro-beta,Gro-gamma, ENA-78, and GCP-2; in other words the penta-specific antibodyof the present invention minimally binds to human IL-8, Gro-alpha,Gro-beta, Gro-gamma, and ENA-78. Moreover, an isolated penta-specificantibody is substantially free of other cellular material and/orchemicals. A penta-specific antibody, as used in this context, alsoincludes antigen binding fragments and/or derivatives having the bindingcharacteristics of their “parent” penta-specific antibody. Thepenta-specific antibody of the present invention may comprise twoidentical heavy chains and two identical light chains, forming thetypical, bilaterally symmetric immunoglobulin molecule comprised of twoheterodimers which are each comprised of a heavy chain and a lightchain. Accordingly, the penta-specific antibody of the present inventionmay comprise two copies of the same antigen binding domain formed by theassociation of one heavy chain with one light chain. The penta-specificantibody of the present invention, though having only one kind ofbinding domain is still able to bind to and therefore cross react withhuman IL-8, Gro-alpha, Gro-beta, Gro-gamma, and ENA-78.

As used herein, “antibody” is also referred to as “immunoglobulin”.

As used herein, “an antibody that cross reacts with” means the antibodybinds not only to one antigen but binds to other antigens as well.

“Neutralizing,” as used herein is intended to refer to a partial or fullinhibition of at least one biological activities of human IL-8,Gro-alpha, Gro-beta, Gro-gamma, GCP-2, or ENA-78. For example, one ofthe biological activities of human IL-8, Gro-alpha, Gro-beta, Gro-gamma,GCP-2, or ENA-78 is its ability to induce neutrophil chemotaxis.

One way of measuring the binding kinetics of an antibody is by surfaceplasmon resonance. The term “surface plasmon resonance”, as used herein,refers to an optical phenomenon that allows for the analysis ofreal-time biospecific interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277. The term“epitope” means a protein determinant capable of specific binding to anantibody. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

A “monoclonal antibody” or mAb (as opposed to polyclonal antibody) asused herein is intended to refer to a preparation of antibody moleculesof single molecular composition. For example, a murine derivedmonoclonal antibody (mouse monoclonal antibody) can be prepared byhybridoma technology, such as the standard Kohler and Milstein hybridomamethodology.

Antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975,Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75).The technology for producing monoclonal antibody hybridomas is wellknown (see generally R. H. Kenneth, in Monoclonal Antibodies: A NewDimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L.Gefter et al. (1977) Somatic Cell Genet., 3:231-36).

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293cells, plant cells, or fungi, including yeast cells.

As used herein, “specific” binding refers to antibody binding to apredetermined antigen. Typically, the antibody binds with an equilibriumconstant, KD, corresponding to about 1×10⁻⁷ M or less, and binds to thepredetermined antigen with an affinity corresponding to a KD that is atleast two orders of magnitude lower than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen. The phrases “an antibodyrecognizing an antigen” and “an antibody specific for an antigen” areused interchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

As used herein, the term “kd” (sec-1), as used herein, is intended torefer to the dissociation rate constant of a particular antibody-antigeninteraction.

The term “ka” (M×sec-1), as used herein, is intended to refer to theassociation rate constant of a particular antibody-antigen interaction.

The term “KD” (M), as used herein, is intended to refer to theequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the kd by the ka.

“Conservative sequence modifications” for nucleotide and amino acidsequence modifications means changes which do not significantly affector alter the binding characteristics of the antibody encoded by thenucleotide sequence or containing the amino acid sequence. Suchconservative sequence modifications include nucleotide and amino acidsubstitutions, additions and deletions. Modifications can be introducedinto the sequences by standard techniques known in the art, such assite-directed mutagenesis and PCR-mediated mutagenesis. Conservativeamino acid substitutions include ones in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an antibody for whichsequence is specifically disclosed is preferably replaced with anotheramino acid residue from the same side chain family. Thus in one aspect,the penta-specific antibody of the present invention includes all theconservative sequence modifications of the specifically disclosed aminoacid sequences.

The present invention also encompasses “derivatives” of the amino acidsequences as specifically disclosed, wherein one or more of the aminoacid residues have been derivatized, e.g., by acylation orglycosylation, without significantly affecting or altering the bindingcharacteristics of the antibody containing the amino acid sequences.

For nucleic acids, the term “substantial identity” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial identity when the segments willhybridize under selective hybridization conditions, to the complement ofthe strand.

For nucleotide and amino acid sequences, the term “identity” indicatesthe degree of identity between two nucleic acid or amino acid sequenceswhen optimally aligned and compared with appropriate insertions ordeletions. Alternatively, substantial identity exists when the DNAsegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions times 100), taking into accountthe number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide or polypeptide sequences canbe determined using the GAP program in the GCG software package(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. The percent identity between two nucleotide or amino acidsequences can also be determined using the algorithm of E. Meyers and W.Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package (available at http://www.gcg.com), using eithera Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionof regulatory sequences, operably linked means that the DNA sequencesbeing linked are contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame. For switch sequences,operably linked indicates that the sequences are capable of effectingswitch recombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, transfectomas,such as CHO cells, NS/0 cells, and lymphocytic cells.

As used herein, the term “subject” includes any human or non-humananimal. The term “non-human animal” includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

1. Antibody Structures Intact Antibodies

Intact antibodies are usually heteromultimeric glycoproteins comprisingat least two heavy and two light chains. Aside from IgM, intactantibodies are heterotetrameric glycoproteins of approximately 150 Kda,composed of two identical light (L) chains and two identical heavy (H)chains. Typically, each light chain is linked to a heavy chain by onecovalent disulfide bond while the number of disulfide linkages betweenthe heavy chains of different immunoglobulin isotypes varies. Each heavyand light chain also has intrachain disulfide bridges. Each heavy chainhas at one end a variable domain (V_(H)) followed by a number ofconstant regions. Each light chain has a variable domain (V_(L)) and aconstant region at its other end; the constant region of the light chainis aligned with the first constant region of the heavy chain and thelight chain variable domain is aligned with the variable domain of theheavy chain. The light chains of antibodies from most vertebrate speciescan be assigned to one of two types called Kappa and Lambda based on theamino acid sequence of the constant region. Depending on the amino acidsequence of the constant region of their heavy chains, human antibodiescan be assigned to five different classes, IgA, IgD, IgE, IgG and IgM.IgG and IgA can be further subdivided into subclasses, IgG1, IgG2, IgG3and IgG4; and IgA1 and IgA2. Species variants exist with mouse and rathaving at least IgG2a, IgG2b. The variable domain of the antibodyconfers binding specificity upon the antibody with certain regionsdisplaying particular variability called complementarity determiningregions (CDRs). The more conserved portions of the variable region arecalled framework regions (FR). The variable domains of intact heavy andlight chains each comprise four FR connected by three CDRs. The CDRs ineach chain are held together in close proximity by the FR regions andwith the CDRs from the other chain contribute to the formation of theantigen binding site of antibodies. The constant regions are notdirectly involved in the binding of the antibody to the antigen butexhibit various effector functions such as participation in antibodydependent cell-mediated cytotoxicity (ADCC), phagocytosis via binding toFcγ receptor, half-life/clearance rate via neonatal Fc receptor (FcRn)and complement dependent cytotoxicity via the C1q component of thecomplement cascade. The human IgG2 constant region lacks the ability toactivate complement by the classical pathway or to mediateantibody-dependent cellular cytotoxicity. The IgG4 constant region lacksthe ability to activate complement by the classical pathway and mediatesantibody-dependent cellular cytotoxicity only weakly. Antibodiesessentially lacking these effector functions may be termed ‘non-lytic’antibodies.

Human Antibodies

Human antibodies may be produced by a number of methods known to thoseof skill in the art. Human antibodies can be made by the hybridomamethod using human myeloma or mouse-human heteromyeloma cells lines seeKozbor J. Immunol 133, 3001, (1984) and Brodeur, Monoclonal AntibodyProduction Techniques and Applications, pp 51-63 (Marcel Dekker Inc,1987). Alternative methods include the use of phage libraries ortransgenic mice both of which utilize human V region repertories (seeWinter G, (1994), Annu. Rev. Immunol 12, 433-455, Green L L (1999), J.Immunol. methods 231, 11-23).

Several strains of transgenic mice are now available wherein their mouseimmunoglobulin loci has been replaced with human immunoglobulin genesegments (see Tomizuka K, (2000) PNAS 97, 722-727; Fishwild D. M (1996)Nature Biotechnol. 14, 845-851, Mendez M J, 1997, Nature Genetics, 15,146-156). Upon antigen challenge such mice are capable of producing arepertoire of human antibodies from which antibodies of interest can beselected.

Of particular note is the Trimera™ system (see Eren R et al, (1998)Immunology 93:154-161) where human lymphocytes are transplanted intoirradiated mice, the Selected Lymphocyte Antibody System (SLAM, seeBabcook et al, PNAS (1996) 93:7843-7848) where human (or other species)lymphocytes are effectively put through a massive pooled in vitroantibody generation procedure followed by deconvulated, limitingdilution and selection procedure and the Xenomouse II™ (Abgenix Inc). Analternative approach is available from Morphotek Inc using theMorphodoma™ technology.

Phage display technology can be used to produce human antibodies (andfragments thereof), see McCafferty; Nature, 348, 552-553 (1990) andGriffiths A D et al (1994) EMBO 13:3245-3260. According to thistechnique antibody V domain genes are cloned in frame into either amajor or minor coat of protein gene of a filamentous bacteriophage suchas M13 or fd and displayed (usually with the aid of a helper phage) asfunctional antibody fragments on the surface of the phage particle.Selections based on the functional properties of the antibody result inselection of the gene encoding the antibody exhibiting those properties.The phage display technique can be used to select antigen specificantibodies from libraries made from human B cells taken from individualsafflicted with a disease or disorder described above or alternativelyfrom unimmunized human donors (see Marks; J. Mol. Bio. 222, 581-597,1991). Where an intact human antibody is desired comprising a Fc domainit is necessary to reclone the phage displayed derived fragment into amammalian expression vectors comprising the desired constant regions andestablishing stable expressing cell lines.

The technique of affinity maturation (Marks; Bio/technol 10, 779-783(1992)) may be used to improve binding affinity wherein the affinity ofthe primary human antibody is improved by sequentially replacing the Hand L chain V regions with naturally occurring variants and selecting onthe basis of improved binding affinities. Variants of this techniquesuch as “epitope imprinting” are now also available see WO 93/06213. Seealso Waterhouse; Nucl. Acids Res 21, 2265-2266 (1993).

Chimaeric and Humanised Antibodies

The use of intact non-human antibodies in the treatment of humandiseases or disorders carries with it the now well established problemsof potential immunogenicity especially upon repeated administration ofthe antibody that is the immune system of the patient may recognise thenon-human intact antibody as non-self and mount a neutralising response.In addition to developing fully human antibodies (see above) varioustechniques have been developed over the years to overcome these problemsand generally involve reducing the composition of non-human amino acidsequences in the intact therapeutic antibody whilst retaining therelative ease in obtaining non-human antibodies from an immunised animale.g. mouse, rat or rabbit. Broadly two approaches have been used toachieve this. The first are chimaeric antibodies, which generallycomprise a non-human (e.g. rodent such as mouse) variable domain fusedto a human constant region. Because the antigen-binding site of anantibody is localised within the variable regions the chimaeric antibodyretains its binding affinity for the antigen but acquires the effectorfunctions of the human constant region and are therefore able to performeffector functions such as described supra. Chimaeric antibodies aretypically produced using recombinant DNA methods. DNA encoding theantibodies (e.g. cDNA) is isolated and sequenced using conventionalprocedures (e.g. by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the H and L chain variableregions of the antibody of the invention. Hybridoma cells serve as atypical source of such DNA. Once isolated, the DNA is placed intoexpression vectors which are then transfected into host cells such as E.Coli, COS cells, CHO cells or myeloma cells that do not otherwiseproduce immunoglobulin protein to obtain synthesis of the antibody. TheDNA may be modified by substituting the coding sequence for human L andH chains for the corresponding non-human (e.g. murine) H and L constantregions see e.g. Morrison; PNAS 81, 6851 (1984). Thus another embodimentof the invention there is provided a chimaeric antibody comprising aV_(H) domain having the sequence: SEQ ID No:2, 6, or 10 and a V_(L)domain having the sequence: SEQ ID No: 4, 8, or 12 fused to a humanconstant region (which maybe of a IgG isotype e.g. IgG1).

The second approach involves the generation of humanised antibodieswherein the non-human content of the antibody is reduced by humanizingthe variable regions. Two techniques for humanisation have gainedpopularity. The first is humanisation by CDR grafting. CDRs build loopsclose to the antibody's N-terminus where they form a surface mounted ina scaffold provided by the framework regions. Antigen-bindingspecificity of the antibody is mainly defined by the topography and bythe chemical characteristics of its CDR surface. These features are inturn determined by the conformation of the individual CDRs, by therelative disposition of the CDRs, and by the nature and disposition ofthe side chains of the residues comprising the CDRs. A large decrease inimmunogenicity can be achieved by grafting only the CDRs of a non-human(e.g. murine) antibodies (“donor” antibodies) onto a suitable humanframework (“acceptor framework”) and constant regions (see Jones et al(1986) Nature 321, 522-525 and Verhoeyen M et al (1988) Science 239,1534-1536). However, CDR grafting per se may not result in the completeretention of antigen-binding properties and it is frequently found thatsome framework residues of the donor antibody need to be preserved(sometimes referred to as “backmutations”) in the humanised molecule ifsignificant antigen-binding affinity is to be recovered (see Queen C etal (1989) PNAS 86, 10,029-10,033, Co, M et al (1991) Nature 351,501-502). In this case, human V regions showing the greatest sequencehomology (typically 60% or greater) to the non-human donor antibodymaybe chosen from a database in order to provide the human framework(FR). The selection of human FRs can be made either from human consensusor individual human antibodies. Where necessary key residues from thedonor antibody are substituted into the human acceptor framework topreserve CDR conformations. Computer modelling of the antibody maybeused to help identify such structurally important residues, seeWO99/48523.

Alternatively, humanisation maybe achieved by a process of “veneering”.A statistical analysis of unique human and murine immunoglobulin heavyand light chain variable regions revealed that the precise patterns ofexposed residues are different in human and murine antibodies, and mostindividual surface positions have a strong preference for a small numberof different residues (see Padlan E. A. et al; (1991) Mol. Immunol. 28,489-498 and Pedersen J. T. et al (1994) J. Mol. Biol. 235; 959-973).Therefore it is possible to reduce the immunogenicity of a non-human Fvby replacing exposed residues in its framework regions that differ fromthose usually found in human antibodies. Because protein antigenicitycan be correlated with surface accessibility, replacement of the surfaceresidues may be sufficient to render the mouse variable region“invisible” to the human immune system (see also Mark G. E. et al (1994)in Handbook of Experimental Pharmacology vol. 113: The pharmacology ofmonoclonal Antibodies, Springer-Verlag, pp 105-134). This procedure ofhumanisation is referred to as “veneering” because only the surface ofthe antibody is altered, the supporting residues remain undisturbed. Afurther alternative approach is set out in WO04/006955. Furtheralternative approaches include that set out in WO04/006955 and theprocedure of Humaneering™ (Kalobios) which makes use of bacterialexpression systems and produces antibodies that are close to humangermline in sequence (Alfenito-M Advancing Protein Therapeutics January2007, San Diego, Calif.). Another, approach to humanisation involvesselecting human acceptor frameworks on the basis of structuralsimilarity of the human CDR regions to those of the donor mouse antibodyCDR regions rather than on homology between other regions of theantibody such as framework regions. This process is also known asSuperhumanisation™ (Evogenix Inc.; Hwang et al (2005) Methods 36:35-42).

It will be apparent to those skilled in the art that the term “derived”is intended to define not only the source in the sense of it being thephysical origin for the material but also to define material which isstructurally identical to the material but which does not originate fromthe reference source. Thus “residues found in the donor antibody” neednot necessarily have been purified from the donor antibody.

Antibody Fragments

In certain embodiments of the invention there is provided therapeuticantibody which is an antigen binding fragment. Such fragments may befunctional antigen binding fragments of intact and/or humanised and/orchimaeric antibodies such as Fab, Fd, Fab′, F(ab′)₂, Fv, ScFv fragmentsof the antibodies described supra. Fragments lacking the constant regionlack the ability to activate complement by the classical pathway or tomediate antibody-dependent cellular cytotoxicity. Traditionally suchfragments are produced by the proteolytic digestion of intact antibodiesby e.g. papain digestion (see for example, WO 94/29348) but may beproduced directly from recombinantly transformed host cells. For theproduction of ScFv, see Bird et al; (1988) Science, 242, 423-426. Inaddition, antibody fragments may be produced using a variety ofengineering techniques as described below.

Fv fragments appear to have lower interaction energy of their two chainsthan Fab fragments. To stabilise the association of the V_(H) and V_(L)domains, they have been linked with peptides (Bird et al, (1988) Science242, 423-426, Huston et al, PNAS, 85, 5879-5883), disulphide bridges(Glockshuber et al, (1990) Biochemistry, 29, 1362-1367) and “knob inhole” mutations (Zhu et al (1997), Protein Sci., 6, 781-788). ScFvfragments can be produced by methods well known to those skilled in theart see Whitlow et al (1991) Methods companion Methods Enzymol, 2,97-105 and Huston et al (1993) Int. Rev. Immunol 10, 195-217. ScFv maybe produced in bacterial cells such as E. Coli but are more typicallyproduced in eukaryotic cells. One disadvantage of ScFv is themonovalency of the product, which precludes an increased avidity due topolyvalent binding, and their short half-life. Attempts to overcomethese problems include bivalent (ScFv′)₂ produced from ScFV containingan additional C terminal cysteine by chemical coupling (Adams et al(1993) Can. Res 53, 4026-4034 and McCartney et al (1995) Protein Eng. 8,301-314) or by spontaneous site-specific dimerization of ScFv containingan unpaired C terminal cysteine residue (see Kipriyanov et al (1995)Cell. Biophys 26, 187-204). Alternatively, ScFv can be forced to formmultimers by shortening the peptide linker to between 3 to 12 residuesto form “diabodies”, see Holliger et al PNAS (1993), 90, 6444-6448.Reducing the linker still further can result in ScFV trimers(“triabodies”, see Kortt et al (1997) Protein Eng, 10, 423-433) andtetramers (“tetrabodies”, see Le Gall et al (1999) FEBS Lett, 453,164-168). Construction of bivalent ScFV molecules can also be achievedby genetic fusion with protein dimerizing motifs to form“miniantibodies” (see Pack et al (1992) Biochemistry 31, 1579-1584) and“minibodies” (see Hu et al (1996), Cancer Res. 56, 3055-3061).ScFv-Sc-Fv tandems ((ScFV)₂) may also be produced by linking two ScFvunits by a third peptide linker, see Kurucz et al (1995) J. Immol. 154,4576-4582. Bispecific diabodies can be produced through the noncovalentassociation of two single chain fusion products consisting of V_(H)domain from one antibody connected by a short linker to the V_(L) domainof another antibody, see Kipriyanov et al (1998), Int. J. Can 77,763-772. The stability of such bispecific diabodies can be enhanced bythe introduction of disulphide bridges or “knob in hole” mutations asdescribed supra or by the formation of single chain diabodies (ScDb)wherein two hybrid ScFv fragments are connected through a peptide linkersee Kontermann et al (1999) J. Immunol. Methods 226 179-188. Tetravalentbispecific molecules are available by e.g. fusing a ScFv fragment to theCH3 domain of an IgG molecule or to a Fab fragment through the hingeregion see Coloma et al (1997) Nature Biotechnol. 15, 159-163.Alternatively, tetravalent bispecific molecules have been created by thefusion of bispecific single chain diabodies (see Alt et al, (1999) FEBSLett 454, 90-94. Smaller tetravalent bispecific molecules can also beformed by the dimerization of either ScFv-ScFv tandems with a linkercontaining a helix-loop-helix motif (DiBi miniantibodies, see Muller etal (1998) FEBS Lett 432, 45-49) or a single chain molecule comprisingfour antibody variable domains (V_(H) and V_(L)) in an orientationpreventing intramolecular pairing (tandem diabody, see Kipriyanov et al,(1999) J. Mol. Biol. 293, 41-56). Bispecific F(ab′)2 fragments can becreated by chemical coupling of Fab′ fragments or by heterodimerizationthrough leucine zippers (see Shalaby et al, (1992) J. Exp. Med. 175,217-225 and Kostelny et al (1992), J. Immunol. 148, 1547-1553). Alsoavailable are so-called domain antibodies based on isolated V_(H) orV_(L) domains (Domantis Ltd.), see U.S. Pat. No. 6,248,516; U.S. Pat.No. 6,291,158; U.S. Pat. No. 6,172,197.

Heteroconjugate Antibodies

Heteroconjugate antibodies are derivatives which also form an embodimentof the present invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies formed using any convenient cross-linkingmethods. See U.S. Pat. No. 4,676,980.

Other Modifications.

Antibodies of the present invention may also incorporate any othermodifications in the constant regions. For example glycosylation ofantibodies at conserved positions in their constant regions is known tohave a profound effect on antibody function, particularly effectorfunctioning such as those described above, see for example, Boyd et al(1996), Mol. Immunol. 32, 1311-1318. Glycosylation variants of thetherapeutic antibodies of the present invention wherein one or morecarbohydrate moiety is added, substituted, deleted or modified arecontemplated. Introduction of an asparagine-X-serine orasparagine-X-threonine motif creates a potential site for enzymaticattachment of carbonhydrate moieties and may therefore be used tomanipulate the glycosylation of an antibody. In Raju et al (2001)Biochemistry 40, 8868-8876 the terminal sialyation of a TNFR-IgGimmunoadhesin was increased through a process of regalactosylationand/or resialylation using beta-1,4-galactosyltransferace and/or alpha,2,3 sialyltransferase. Increasing the terminal sialylation is believedto increase the half-life of the immunoglobulin. Antibodies, in commonwith most glycoproteins, are typically produced in nature as a mixtureof glycoforms. This mixture is particularly apparent when antibodies areproduced in eukaryotic, particularly mammalian cells. A variety ofmethods have been developed to manufacture defined glycoforms, see Zhanget al Science (2004), 303, 371, Sears et al, Science, (2001) 291, 2344,Wacker et al (2002) Science, 298 1790, Davis et al (2002) Chem. Rev.102, 579, Hang et al (2001) Acc. Chem. Res 34, 727. Thus the inventionconcerns a plurality of therapeutic antibodies (which maybe of the IgGisotype, e.g. IgG1) as described herein comprising a defined number(e.g. 7 or less, for example 5 or less such as two or a single)glycoform(s) of said antibodies.

Derivatives according to the invention also include therapeuticantibodies of the invention coupled to a non-proteinaeous polymer suchas polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.Conjugation of proteins to PEG is an established technique forincreasing half-life of proteins, as well as reducing antigenicity andimmunogenicity of proteins. The use of PEGylation with differentmolecular weights and styles (linear or branched) has been investigatedwith intact antibodies as well as Fab′ fragments, see Koumenis I. L. etal (2000) Int. J. Pharmaceut. 198:83-95. A particular embodimentcomprises an antigen-binding fragment of the invention without theeffector functions of a) activation of complement by the classicalpathway; and b) mediating antibody-dependent cellular cytotoxicity;(such as a Fab fragment or a scFv) coupled to PEG.

The interaction between the Fc region of an antibody and various Fcreceptors (FcγR) is believed to mediate the effector functions of theantibody which include antibody-dependent cellular cytotoxicity (ADCC),fixation of complement, phagocytosis and half-life/clearance of theantibody. Various modifications to the Fc region of antibodies of theinvention may be carried out depending on the desired effector property.In particular, human constant regions which essentially lack thefunctions of a) activation of complement by the classical pathway; andb) mediating antibody-dependent cellular cytotoxicity include the IgG4constant region, the IgG2 constant region and IgG1 constant regionscontaining specific mutations as for example mutations at positions 234,235, 236, 237, 297, 318, 320 and/or 322 disclosed in EP0307434(WO8807089), EP 0629 240 (WO9317105) and WO 2004/014953. Mutations atresidues 235 or 237 within the CH2 domain of the heavy chain constantregion (Kabat numbering; EU Index system) have separately been describedto reduce binding to FcγRI, FcγRII and FcγRIII binding and thereforereduce antibody-dependent cellular cytotoxicity (ADCC) (Duncan et al.Nature 1988, 332; 563-564; Lund et al. J. Immunol. 1991, 147; 2657-2662;Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. Immunol.1992, 51; 1-84; Morgan et al., Immunology 1995, 86; 319-324; Hezareh etal., J. Virol. 2001, 75 (24); 12161-12168). Further, some reports havealso described involvement of some of these residues in recruiting ormediating complement dependent cytotoxicity (CDC) (Morgan et al., 1995;Xu et al., Cell. Immunol. 2000; 200:16-26; Hezareh et al., J. Virol.2001, 75 (24); 12161-12168). Residues 235 and 237 have therefore bothbeen mutated to alanine residues (Brett et al. Immunology 1997, 91;346-353; Bartholomew et al. Immunology 1995, 85; 41-48; and WO9958679)to reduce both complement mediated and FcγR-mediated effects. Antibodiescomprising these constant regions may be termed ‘non-lytic’ antibodies.

One may incorporate a salvage receptor binding epitope into the antibodyto increase serum half life see U.S. Pat. No. 5,739,277.

There are five currently recognised human Fcγ receptors, FcγR (I),FcγRIIa, FcγRIIb, FcγRIIIa and neonatal FcRn. Shields et al, (2001) J.Biol. Chem 276, 6591-6604 demonstrated that a common set of IgG1residues is involved in binding all FcγRs, while FcγRII and FcγRIIIutilize distinct sites outside of this common set. One group of IgG1residues reduced binding to all FcγRs when altered to alanine: Pro-238,Asp-265, Asp-270, Asn-297 and Pro-239. All are in the IgG CH2 domain andclustered near the hinge joining CH1 and CH2. While FcγRI utilizes onlythe common set of IgG1 residues for binding, FcγRII and FcγRIII interactwith distinct residues in addition to the common set. Alteration of someresidues reduced binding only to FcγRII (e.g. Arg-292) or FcγRIII (e.g.Glu-293). Some variants showed improved binding to FcγRII or FcγRIII butdid not affect binding to the other receptor (e.g. Ser-267Ala improvedbinding to FcγRII but binding to FcγRIII was unaffected). Other variantsexhibited improved binding to FcγRII or FcγRIII with reduction inbinding to the other receptor (e.g. Ser-298Ala improved binding toFcγRIII and reduced binding to FcγRII). For FcγRIIIa, the best bindingIgG1 variants had combined alanine substitutions at Ser-298, Glu-333 andLys-334. The neonatal FcRn receptor is believed to be involved inprotecting IgG molecules from degradation and thus enhancing serum halflife and the transcytosis across tissues (see Junghans R. P (1997)Immunol. Res 16. 29-57 and Ghetie et al (2000) Annu. Rev. Immunol. 18,739-766). Human IgG1 residues determined to interact directly with humanFcRn includes Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435.

The therapeutic antibody of the invention may incorporate any of theabove constant region modifications.

2. Production Methods

Antibodies of the present invention may be produced in transgenicorganisms such as goats (see Pollock et al (1999), J. Immunol. Methods231:147-157), chickens (see Morrow K J J (2000) Genet. Eng. News20:1-55), mice (see Pollock et al ibid) or plants (see Doran P M, (2000)Curr. Opinion Biotechnol. 11, 199-204, Ma J K-C (1998), Nat. Med. 4;601-606, Baez J et al, BioPharm (2000) 13: 50-54, Stoger E et al; (2000)Plant Mol. Biol. 42:583-590). Antibodies may also be produced bychemical synthesis. However, antibodies of the invention are typicallyproduced using recombinant cell culturing technology well known to thoseskilled in the art. A polynucleotide encoding the antibody is isolatedand inserted into a replicable vector such as a plasmid for furthercloning (amplification) or expression in a host cell. One usefulexpression system is a glutamate synthetase system (such as sold byLonza Biologics), particularly where the host cell is CHO or NS0 (seebelow). Polynucleotide encoding the antibody is readily isolated andsequenced using conventional procedures (e.g. oligonucleotide probes).Vectors that may be used include plasmid, virus, phage, transposons,minichromsomes of which plasmids are a typical embodiment. Generallysuch vectors further include a signal sequence, origin of replication,one or more marker genes, an enhancer element, a promoter andtranscription termination sequences operably linked to the light and/orheavy chain polynucleotide so as to facilitate expression.Polynucleotide encoding the light and heavy chains may be inserted intoseparate vectors and introduced (e.g. by transformation, transfection,electroporation or transduction) into the same host cell concurrently orsequentially or, if desired both the heavy chain and light chain can beinserted into the same vector prior to such introduction.

It will be immediately apparent to those skilled in the art that due tothe redundancy of the genetic code, alternative polynucleotides to thosedisclosed herein are also available that will encode the polypeptides ofthe invention.

Signal Sequences

Antibodies of the present invention maybe produced as a fusion proteinwith a heterologous signal sequence having a specific cleavage site atthe N terminus of the mature protein. The signal sequence should berecognised and processed by the host cell. For prokaryotic host cells,the signal sequence may be an alkaline phosphatase, penicillinase, orheat stable enterotoxin II leaders. For yeast secretion the signalsequences may be a yeast invertase leader, a factor leader or acidphosphatase leaders see e.g. WO90/13646. In mammalian cell systems,viral secretory leaders such as herpes simplex gD signal and a nativeimmunoglobulin signal sequence (such as human Ig heavy chain) areavailable. Typically the signal sequence is ligated in reading frame topolynucleotide encoding the antibody of the invention.

Origin of Replication

Origin of replications are well known in the art with pBR322 suitablefor most gram-negative bacteria, 2μ plasmid for most yeast and variousviral origins such as SV40, polyoma, adenovirus, VSV or BPV for mostmammalian cells. Generally the origin of replication component is notneeded for integrated mammalian expression vectors, unless vectorpropagation is required in E. Coli. However the SV40 ori may be usedsince it contains the early promoter.

Selection Marker

Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins e.g. ampicillin, neomycin, methotrexate ortetracycline or (b) complement auxotrophic deficiencies or supplynutrients not available in the complex media or (c) combinations ofboth. The selection scheme may involve arresting growth of the hostcells that contain no vector or vectors. Cells, which have beensuccessfully transformed with the genes encoding the therapeuticantibody of the present invention, survive due to e.g. drug resistanceconferred by the co-delivered selection marker. One example is theDHFR-selection system wherein transformants are generated in DHFRnegative host strains (eg see Page and Sydenham 1991 Biotechnology 9:64-68). In this system the DHFR gene is co-delivered with antibodypolynucleotide sequences of the invention and DHFR positive cells thenselected by nucleoside withdrawal. If required, the DHFR inhibitormethotrexate is also employed to select for transformants with DHFR geneamplification. By operably linking DHFR gene to the antibody codingsequences of the invention or functional derivatives thereof, DHFR geneamplification results in concomitant amplification of the desiredantibody sequences of interest. CHO cells are a particularly useful cellline for this DHFR/methotrexate selection and methods of amplifying andselecting host cells using the DHFR system are well established in theart see Kaufman R. J. et al J. Mol. Biol. (1982) 159, 601-621, forreview, see Werner R G, Noe W, Kopp K, Schluter M, “Appropriatemammalian expression systems for biopharmaceuticals”,Arzneimittel-Forschung. 48(8):870-80, 1998 Aug. A further example is theglutamate synthetase expression system (Lonza Biologics). A suitableselection gene for use in yeast is the trp1 gene; see Stinchcomb et alNature 282, 38, 1979.

Promoters

Suitable promoters for expressing antibodies of the invention areoperably linked to DNA/polynucleotide encoding the antibody. Promotersfor prokaryotic hosts include phoA promoter, Beta-lactamase and lactosepromoter systems, alkaline phosphatase, tryptophan and hybrid promoterssuch as Tac. Promoters suitable for expression in yeast cells include3-phosphoglycerate kinase or other glycolytic enzymes e.g. enolase,glyceralderhyde 3 phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose 6 phosphate isomerase,3-phosphoglycerate mutase and glucokinase, among others. Inducible yeastpromoters include alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, metallothionein and enzymes responsible for nitrogenmetabolism or maltose/galactose utilization, among others. Promoters forexpression in mammalian cell systems include RNA polymerase II promotersincluding viral promoters such as polyoma, fowlpox and adenoviruses(e.g. adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus (in particular the immediate early gene promoter),retrovirus, hepatitis B virus, actin, rous sarcoma virus (RSV) promoterand the early or late Simian virus 40 and non-viral promoters such asEF-1alpha (Mizushima and Nagata Nucleic Acids Res 1990 18(17):5322,among others. The choice of promoter may be based upon suitablecompatibility with the host cell used for expression.

Enhancer Element

Where appropriate, e.g. for expression in higher eukaroytics, additionalenhancer elements can included instead of or as well as those foundlocated in the promoters described above. Suitable mammalian enhancersequences include enhancer elements from globin, elastase, albumin,fetoprotein, metallothionine and insulin. Alternatively, one may use anenhancer element from a eukaroytic cell virus such as SV40 enhancer,cytomegalovirus early promoter enhancer, polyoma enhancer, baculoviralenhancer or murine IgG2a locus (see WO04/009823). Whilst such enhancersare typically located on the vector at a site upstream to the promoter,they can also be located elsewhere e.g. within the untranslated regionor downstream of the polydenalytion signal. The choice and positioningof enhancer may be based upon suitable compatibility with the host cellused for expression.

Polyadenylation/Termination

In eukaryotic systems, polyadenylation signals are operably linked topolynucleotide encoding the antibody of this invention. Such signals aretypically placed 3′ of the open reading frame. In mammalian systems,non-limiting example signals include those derived from growth hormones,elongation factor-1 alpha and viral (eg SV40) genes or retroviral longterminal repeats. In yeast systems non-limiting examples ofpolydenylation/termination signals include those derived from thephosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 (ADH)genes. In prokaryotic system polyadenylation signals are typically notrequired and it is instead usual to employ shorter and more definedterminator sequences. The choice of polyadenylation/terminationsequences may be based upon suitable compatibility with the host cellused for expression.

Other Methods/Elements for Enhanced Yields

In addition to the above, other features that can be employed to enhanceyields include chromatin remodelling elements, introns and host-cellspecific codon modification. The codon usage of the antibody of thisinvention thereof can be modified to accommodate codon bias of the hostcell such to augment transcript and/or product yield (eg Hoekema A et alMol Cell Biol 1987 7(8):2914-24). The choice of codons may be based uponsuitable compatibility with the host cell used for expression.

Host Cells

Suitable host cells for cloning or expressing vectors encodingantibodies of the invention are, for example, prokaroytic, yeast orhigher eukaryotic cells. Suitable prokaryotic cells include eubacteriae.g. enterobacteriaceae such as Escherichia e.g. E. Coli (for exampleATCC 31,446; 31,537; 27,325), Enterobacter, Erwinia, Klebsiella Proteus,Salmonella e.g. Salmonella typhimurium, Serratia e.g. Serratiamarcescans and Shigella as well as Bacilli such as B. subtilis and B.licheniformis (see DD 266 710), Pseudomonas such as P. aeruginosa andStreptomyces. Of the yeast host cells, Saccharomyces cerevisiae,schizosaccharomyces pombe, Kluyveromyces (e.g. ATCC 16,045; 12,424;24178; 56,500), yarrowia (EP402, 226), Pichia Pastoris (EP183, 070, seealso Peng et al J. Biotechnol. 108 (2004) 185-192), Candida, Trichodermareesia (EP244, 234), Penicillin, Tolypocladium and Aspergillus hostssuch as A. nidulans and A. niger are also contemplated.

Although Prokaryotic and yeast host cells are specifically contemplatedby the invention, typically however, host cells of the present inventionare vertebrate cells. Suitable vertebrate host cells include mammaliancells such as COS-1 (ATCC No. CRL 1650) COS-7 (ATCC CRL 1651), humanembryonic kidney line 293, PerC6 (Crucell), baby hamster kidney cells(BHK) (ATCC CRL. 1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCC NO. CRL1573), Chinese hamster ovary cells CHO (e.g. CHO-K1, ATCC NO: CCL 61,DHFR-CHO cell line such as DG44 (see Urlaub et al, (1986) ibid),particularly those CHO cell lines adapted for suspension culture, mousesertoli cells, monkey kidney cells, African green monkey kidney cells(ATCC CRL-1587), HELA cells, canine kidney cells (ATCC CCL 34), humanlung cells (ATCC CCL 75), Hep G2 and myeloma or lymphoma cells e.g. NS0(see U.S. Pat. No. 5,807,715), Sp2/0, Y0.

Thus in one embodiment of the invention there is provided a stablytransformed host cell comprising a vector encoding a heavy chain and/orlight chain of the therapeutic antibody as described herein. Typicallysuch host cells comprise a first vector encoding the light chain and asecond vector encoding said heavy chain.

Such host cells may also be further engineered or adapted to modifyquality, function and/or yield of the antibody of this invention.Non-limiting examples include expression of specific modifying (egglycosylation) enzymes and protein folding chaperones.

Cell Culturing Methods.

Host cells transformed with vectors encoding the therapeutic antibodiesof the invention may be cultured by any method known to those skilled inthe art. Host cells may be cultured in spinner flasks, shake flasks,roller bottles or hollow fibre systems but it is preferred for largescale production that stirred tank reactors or bag reactors (eg WaveBiotech, Somerset, N.J. USA) are used particularly for suspensioncultures. Typically the stirred tankers are adapted for aeration usinge.g. spargers, baffles or low shear impellers. For bubble columns andairlift reactors direct aeration with air or oxygen bubbles maybe used.Where the host cells are cultured in a serum free culture media it ispreferred that the media is supplemented with a cell protective agentsuch as pluronic F-68 to help prevent cell damage as a result of theaeration process. Depending on the host cell characteristics, eithermicrocarriers maybe used as growth substrates for anchorage dependentcell lines or the cells maybe adapted to suspension culture (which istypical). The culturing of host cells, particularly vertebrate hostcells may utilise a variety of operational modes such as batch,fed-batch, repeated batch processing (see Drapeau et al (1994)cytotechnology 15: 103-109), extended batch process or perfusionculture. Although recombinantly transformed mammalian host cells may becultured in serum-containing media such media comprising fetal calfserum (FCS), it is preferred that such host cells are cultured insynthetic serum-free media such as disclosed in Keen et al (1995)Cytotechnology 17:153-163, or commercially available media such asProCHO-CDM or UltraCHO™ (Cambrex N.J., USA), supplemented wherenecessary with an energy source such as glucose and synthetic growthfactors such as recombinant insulin. The serum-free culturing of hostcells may require that those cells are adapted to grow in serum freeconditions. One adaptation approach is to culture such host cells inserum containing media and repeatedly exchange 80% of the culture mediumfor the serum-free media so that the host cells learn to adapt in serumfree conditions (see e.g. Scharfenberg K et al (1995) in Animal Celltechnology: Developments towards the 21st century (Beuvery E. C. et aleds), pp 619-623, Kluwer Academic publishers).

Antibodies of the invention secreted into the media may be recovered andpurified from the media using a variety of techniques to provide adegree of purification suitable for the intended use. For example theuse of therapeutic antibodies of the invention for the treatment ofhuman patients typically mandates at least 95% purity as determined byreducing SDS-PAGE, more typically 98% or 99% purity, when compared tothe culture media comprising the therapeutic antibodies. In the firstinstance, cell debris from the culture media is typically removed usingcentrifugation followed by a clarification step of the supernatant usinge.g. microfiltration, ultrafiltration and/or depth filtration.Alternatively, the antibody can be harvested by microfiltration,ultrafiltration or depth filtration without prior centrifugation. Avariety of other techniques such as dialysis and gel electrophoresis andchromatographic techniques such as hydroxyapatite (HA), affinitychromatography (optionally involving an affinity tagging system such aspolyhistidine) and/or hydrophobic interaction chromatography (HIC, seeU.S. Pat. No. 5,429,746) are available. In one embodiment, theantibodies of the invention, following various clarification steps, arecaptured using Protein A or G affinity chromatography followed byfurther chromatography steps such as ion exchange and/or HAchromatography, anion or cation exchange, size exclusion chromatographyand ammonium sulphate precipitation. Typically, various virus removalsteps are also employed (e.g. nanofiltration using e.g. a DV-20 filter).Following these various steps, a purified (typically monoclonal)preparation comprising at least 10 mg/ml or greater e.g. 100 mg/ml orgreater of the antibody of the invention is provided and therefore formsan embodiment of the invention. Concentration to 100 mg/ml or greatercan be generated by ultracentrifugation. Suitably such preparations aresubstantially free of aggregated forms of antibodies of the invention.

Bacterial systems are particularly suited for the expression of antibodyfragments. Such fragments are localised intracellularly or within theperiplasma. Insoluble periplasmic proteins can be extracted and refoldedto form active proteins according to methods known to those skilled inthe art, see Sanchez et al (1999) J. Biotechnol. 72, 13-20 and Cupit P Met al (1999) Lett Appl Microbiol, 29, 273-277.

3. Pharmaceutical Compositions and Mode of Administration

Purified preparations of antibodies of the invention as described supra,may be incorporated into pharmaceutical compositions for use in thetreatment of human diseases and disorders such as those outlined above.Typically such compositions further comprise a pharmaceuticallyacceptable (i.e. inert) carrier as known and called for by acceptablepharmaceutical practice, see e.g. Remingtons Pharmaceutical Sciences,16th ed, (1980), Mack Publishing Co. Examples of such carriers includesterilised carrier such as saline, Ringers solution or dextrosesolution, buffered with suitable buffers to a pH within a range of 5 to8. Pharmaceutical compositions for injection (e.g. by intravenous,intraperitoneal, intradermal, subcutaneous, intramuscular orintraportal) or continuous infusion are suitably free of visibleparticulate matter and may comprise from 0.1 mg to 10 g of therapeuticantibody, typically between 5 mg and 25 mg of antibody. Methods for thepreparation of such pharmaceutical compositions are well known to thoseskilled in the art. In one embodiment, pharmaceutical compositionscomprise from 0.1 mg to 10 g of therapeutic antibodies of the inventionin unit dosage form, optionally together with instructions for use.Pharmaceutical compositions of the invention may be lyophilised (freezedried) for reconstitution prior to administration according to methodswell known or apparent to those skilled in the art. Where embodiments ofthe invention comprise antibodies of the invention with an IgG1 isotype,a chelator of copper such as citrate (e.g. sodium citrate) or EDTA orhistidine may be added to the pharmaceutical composition to reduce thedegree of copper-mediated degradation of antibodies of this isotype, seeEP0612251.

Effective doses and treatment regimes for administering the antibody ofthe invention are generally determined empirically and are dependent onfactors such as the age, weight and health status of the patient anddisease or disorder to be treated. Such factors are within the purviewof the attending physician. Guidance in selecting appropriate doses maybe found in e.g. Smith et al (1977) Antibodies in human diagnosis andtherapy, Raven Press, New York but will in general be between 0.1 mg and1 g. In one embodiment, the dosing regime for treating a human patientis 0.1 mg to 10 g of therapeutic antibody of the invention administeredsubcutaneously once per week or every two weeks, or by intravenousinfusion every 1 or 2 months. Compositions of the present invention mayalso be used in prophylatically.

4. Clinical Uses.

The present invention relates to an antibody which binds to human IL-8,Gro-alpha, Gro-beta, Gro-gamma, and ENA-78 The present invention alsoconcerns methods of treating diseases or disorders characterised byelevated or unbalanced level of one or more of human IL-8, Gro-alpha,Gro-beta, Gro-gamma, and ENA-78, particularly, COPD, osteoarthritis,rheumatoid arthritis, erosive arthritis, asthma, atherosclerosis,inflammatory bowel disease, psoriasis, transplant rejection, gout,cancer, acute lung injury, acute lung disease, sepsis, ARDS, peripheralartery disease, systemic sclerosis, neonatal respiratory distresssyndrome, exacerbation of asthma and COPD, cystic fibrosis, diffusepanbronchiolitis, reperfusion injury, and/or endometriosis with saidantibody, pharmaceutical compositions comprising said antibody andmethods of manufacture.

The present invention also relates to use of an antibody in themanufacture of a medicament for the treatment of diseases or disorderscharacterised by elevated or unbalanced level of one or more of humanIL-8, Gro-alpha, Gro-beta, Gro-gamma, GCP-2 and ENA-78, particularlyCOPD, osteoarthritis, rheumatoid arthritis, erosive arthritis, asthma,atherosclerosis, inflammatory bowel disease, psoriasis, transplantrejection, gout, cancer, acute lung injury, acute lung disease, sepsis,ARDS, peripheral artery disease, systemic sclerosis, neonatalrespiratory distress syndrome, exacerbation of asthma and COPD, cysticfibrosis, diffuse panbronchiolitis, reperfusion injury, orendometriosis. Although the present invention has been describedprincipally in relation to the treatment of human diseases or disorders,the present invention may also have applications in the treatment ofsimilar diseases or disorders in non-human mammals.

SPECIFIC EMBODIMENTS Example 1 Generation of Mouse Monoclonal Antibody656.35, 197.2, and 81.1

Generation of a Penta-Specific mAb Against the Target Chemokines HumanIL-8, Gro-Alpha, Gro-Beta, Gro-Gamma, and ENA-78).

Multiple methods and schemes were used to immunize mice in an attempt togenerate pan-specific mAbs. The generation of pan-specific mAbs weregenerated using various mixtures of multiple antigenic peptides (MAPs)and/or intact target chemokines (IL-8, Gro-α, -β, -γ, and ENA-78) mixedin complete or incomplete Freund's Adjuvant (cFA or iFA), following amodified Repetitive Immunization Multiple Sites (RIMMS) protocol in theSJL/JOrlCrl mouse strain.

MAPs or multiple antigenic peptides serve two functions within theimmunization protocol. First, MAPs allow for a selective multiplepresentation of a known target amino acid sequence. Secondly, there isan increase in mass, due to multiple copies of the sequence linked, forexample, via a lysine core, which also increases the immunogenicity ofthe sequence over that of individual peptides (Francis, J. P., et al.,Immunology, 1991: 73; 249, Schott, M. E., et al., Cell. Immuno.1996:174:199-209, Tam, J. P. Proc. Natl. Acad. Sci. 1988: 85;5409-5413). FIG. 1 is a schematic drawing of a set of MAPs having aminoacid sequences SEQ ID NOs:49-53. A linker in MAPs can be any linkerother than lysines.

General Immunization Time Line:

Two different immunization protocols following the above time lineproduced pan-specific mAbs:

-   -   1. Initial immunization (day 0) consists of multiple        subcutaneous injections (hind quarters, back and neck) of all        target chemokines mixed in cFA (10 μg each). The following 4        boosts consisted of a mixture of all the target chemokines mixed        in iFA (10 μg each). The fifth and all subsequent boosts        consisted of a cocktail of all the target chemokines and all 5        linear MAPs (10 μg each) in iFA. The final boost 3 days prior to        sacrifice and fusion consisted of all the target chemokines and        linear MAPs in PBS and was delivered via an intraperitoneal (IP)        injection.    -   2. Initial immunization (day 0) consists of multiple        subcutaneous injections (hind quarters, back and neck) of all        five linear MAPs mixed in cFA (10 μg each). The following 4        boosts consisted of a mixture of all five linear MAPs in iFA (10        μg each). The fifth and all subsequent boosts consisted of a        cocktail of all the all 5 linear MAPs and all target chemokines        (10 μg each) in iFA. The final boost 3 days prior to sacrifice        and fusion consisted of all the all 5 linear MAPs and all target        chemokines (10 μg each) in PBS and was delivered via an        intraperitoneal (IP) injection.

Example 2

Pan-binding of the mAb to the target chemokines (human IL-8, Gro-alpha,Gro-beta, Gro-gamma, GCP-2, and ENA-78) has been confirmed via atime-resolved fluorescence immunofluorescent assay (TRFIA).

Briefly, each antigen (human IL-8, Gro-alpha, Gro-beta, Gro-gamma,GCP-2, and ENA-78, and hIL-18 when used as negative control) isindividually coated onto a 96-well immunofluorescent plate. The platesare then washed and blocked with a commercially available blockingsolution. Blocking solution is emptied and samples containing the mAbare added into each well and incubated for 40 to 60 minutes (this allowsthe mAb to bind to the target chemokines). The plates are then washedagain and a detection Ab is added to each well. Detection reagents wereas follows for 2×656.35, chimera (HcLc) and humanized MAbs respectively:biotinylated anti-mouse IgG, biotinylated anti-human IgG Fab2 reagent,and europium anti-human IgG. The detection antibody is conjugated toeither europium or biotin. Following another round of washing, to removeunbound detection Ab, a solution of streptavidin-europium was added tobiotin detected assays. Streptavidin-europium binds to the biotinallowing for detection. After a final wash to remove unboundstreptavidin-europium, or in the case of the humanized MAb assay afterthe europium conjugate secondary is washed, each well is filled with achelating detergent solution to activate the europium producingfluorescence. The greater the fluorescence recorded is directlyproportionate to the amount of detection Ab present in the well which isdirectly linked and proportionate to the amount of the chemokine bindingmAb.FIG. 5A shows mouse 656.35 mAb binding to target human chemokines.FIG. 5B shows the Chimera Antibody (HcLc) mAb binding to human targetchemokines. FIG. 5C shows humanized mAbs binding to human targetchemokines.

Example 3 Functional Pan-Inhibition by Murine mAb was Confirmed Using aVariety of Methods: CXCR2 Mediated Ca²⁺ Mobilization, Human NeutrophilChemotaxis, and Human Neutrophil Activation (CD11b Surface Expression)

A microtiter plate based calcium mobilization assay, FLIPR (FluorometricImaging Plate Reader, Molecular Devices, Sunnyvale Calif., [Schroeder,1996]), was used for the functional characterization of the neutralizingeffect of antibodies on ELR+ chemokine induced [Ca²⁺]i-mobilization inCHO-K1 cells transfected with and stably expressing hCXCR2 and Gα16.

On the day prior to assay, cells were plated in 96 well, blackwall,clear bottom plates (Packard View) at a concentration of 40000 cells perwell. After 18 to 24 hours, media was aspirated off cells and replacedwith 100 μl load media containing Eagles Minimal Essential Medium (EMEM)with Earl's salts and L-Glutamine, 0.1% BSA (Serologicals Corporation),4 μM Fluo-4-acetoxymethyl ester fluorescent indicator dye (Fluo-4 AM)and 2.5 mM probenecid. Cells were incubated in this dye containing mediafor 1 hour at 37° C. The dye containing media was then aspirated off thecells and replaced with identical media without Fluo-4 AM and with 0.1%Gelatin (BSA removed) and 2.5 mM probenecid. Cells were incubated for 10min. at 37° C. and then washed 3 times with KRH assay buffer [KrebsRinger Henseleit (120 mM NaCl, 4.6 mM KCl, 1.03 mM KH₂PO₄, 25 mM NaHCO₃,1.0 mM CaCl₂, 11 mM MgCl₂, 11 mM Glucose, 20 mM HEPES (pH 7.4)) with0.1% gelatin and 2.5 mM probenecid]. After the final buffer wash, 100 μlKRH assay buffer with 0.1% gelatin and 2.5 mM probenecid was added tocells and warmed to 37° C. for 10 min. before being placed in FLIPRwhere dye loaded cells were exposed to excitation light (488 nm) from a6 watt Argon Laser. [Ca²⁺]i-mobilization was monitored as a result of anincrease in 516 nm emission intensity of Fluo-4 when bound to Ca²⁺.Change in emission intensity is directly related to cytosolic calciumlevels, [Ca²⁺]i. After monitoring baseline for 10 sec., 50 μl of 3×ELR+chemokine, which had been pre-incubated with a concentration range ofantibody, was added to the cell plate and data collected every sec. for1 min., followed by an additional half min. of recording in 3 sec.intervals. Maximal cellular response above basal reading was exportedfor plotting in GraphPad Prism (v4.03).

The IC₅₀ was defined as the concentration of antibody required, duringpre-treatment of 3×EC₈₀ chemokine, to neutralize the CXCR2 mediatedstimulatory effect of an EC₈₀ concentration of the ELR+ chemokine by50%. A secondary cellular response to 25 μM ATP was monitored to testcell viability [Sarau, 1999].

TABLE Inhibition of human ELR+ chemokine induced calcium flux(geometrically meaned IC50, ug/ml) by three murine mAb. (n = 3 to 6) 3nM hIL-8 10 nM hGROα 6 nM hGROβ 30 nM hENA-78 100 nM hGCP-2 murine IC₅₀IC₅₀ IC₅₀ IC₅₀ IC₅₀ mAB (ug/ml) 95% C. I. (ug/ml) 95% C. I. (ug/ml) 95%C. I. (ug/ml) 95% C. I. (ug/ml) 95% C. I. 656.35 0.18 0.14-0.25 1.430.60-3.42 1.06 0.78-1.42 2.09 0.71-6.14 9.58 1.17-78.57 81.1 67.65 18.80-243.50 0.94 0.37-2.38 n.d. N/A 1.84 0.69-4.93 9.11 1.44-57.61197.2 2.19 0.61-7.88 2.24  0.39-12.98 n.d. N/A 13.86  1.71-112.49 >157N/A (n.d. = not determined, N/A = not available)Schroeder K S, Neagle, B D. FLIPR: a new instrument for accurate, highthroughput optical screening. J. Biomol. Screen. 1996:1-75.Sarau H M, Ames R S, Chambers J, Ellis C, Elshourbagy N, Foley J J etal. Identification, molecular cloning, expression, and characterizationof a cysteinyl leukotrien receptor. Mol Pharmacol. 1999; 56:657-663.

Inhibition of IL-8 stimulated human neutrophil chemotaxis was alsodemonstrated using a micro-Boyden chamber. The micro-Boyden apparatusconsists of two small chambers one above and one below a 3 micron-porousmembrane. The lower chamber is loaded with the chemotaxic agent (i.e.IL-8) or a mixture of the chemokine with the penta-specific mAb. Theupper chamber contains purified non-activated human neutrophils. Oncethe chamber is assembled a concentration gradient is formed from thebottom well to the upper, stimulating the neutrophils to chemotax acrossthe membrane. The assay is expressed as a CI (chemotactic index) whichis a ratio of the number of cells which chemotax in response to astimulant over non-stimulated chemotaxic cell number. Pre-incubating thechemokine (i.e. IL-8) with the penta-specific mAb in the lower chamber,dose dependently inhibited IL-8 stimulated neutrophil chemotaxis. IL-8at 10 nM achieved a CI of 3.6±0.8. Pre-incubation of IL-8 withincreasing concentrations of the penta-specific mAb (656.35) dosedependently inhibited human neutrophil chemotaxis, significance wasreached at CI of 2.2±0.6 at 1 μg/ml. Due to the amounts and sensitivityof the assay the other chemokines were unable to be examined (Gro-α, -β,-γ, and ENA-78).

Inhibition of purified human neutrophil activation via monitoringsurface expression of CD11b. CD11b or Mac-1 mediates adhesion tosubstrates, aggregation and chemotaxis and is known to be up-regulatedon the surface activated neutrophils (Molad, Y., J., et al., Clin.Immunol. Immunopathol. 1994: 71; 281-286). Briefly, non-activated humanneutrophils are purified and ex vivo stimulated with either targetchemokines (i.e. IL-8) or with chemokines pre-incubated with a chemokinepenta-specific mAb. Data are presented as percent activation set to themaximal CD11b surface expression due to IL-8 stimulation. Pre-incubationof IL-8 with the 656.35 mAb dose dependently inhibited increased levelsof surface expression of CD11b and thus indicates inhibition ofneutrophil activation (79.9%±3.7, 48.5%±7.2, 28.7%±3.2, and 31.6%±3.4,at 0.01, 0.1, 1, 10 and 50 μg/ml respectively).

Example 4 Penta-Specific mAbs Association/Dissociation Values for EachTarget Chemokine (human IL-8, Gro-Beta, and ENA-78) Methods for BiacoreAnalysis.

For experiment designated KL, rabbit anti mouse IgG-Fc (BiacoreBR-1005-14) is immobilized on a CM5 chip by primary amine coupling inaccordance with the manufactures instructions.

For experiment designated BE, purified antibodies directly immobilizedto the chip by amine coupling was used.

Supernatant or purified antibody from parental mouse mAb is captured onthe anti-mouse IgG-Fc surface. Defined concentrations of each analyte(IL8, Gro-band ENA-78) are passed over the immobilized or capturedantibody surface, a separate capture event is used for each analyteinjection. After each injection of analyte the surface is regenerated byinjection

of a mild acidic solution, which removes the captured antibody but doesnot significantly affect the capability anti mouse IgG-Fc surface toperform another capture event nor does it affect the directlyimmobilized antibodies. An injection of buffer is also injected over theantibody captured surface and this is used for double referencing. Thedata is analyzed using the analysis software inherent to the machine,using the 1:1 model of binding.

IL-8 Gro-B ENA-78 mAb kd ka KD kd ka KD kd ka KD KL 656.35 1.48E−031.53E+06 9.64E−10 2.01E−03 3.82E+06 5.26E−10 1.87E−03 1.07E+06 1.74E−09BE 6.87E−04 8.19E+06 8.39E−11 1.46E−03 1.32E+07 1.11E−10 7.96E−042.94E+06 2.71E−10 KL 81.1 9.32E−03 1.95E+06 4.80E−09 3.12E−04 2.76E+061.13E−10 3.76E−04 6.11E+05 6.16E−10 BE 1.08E−01 1.95E+06 5.55E−084.89E−04 5.44E+06 8.98E−11 3.94E−06 1.65E+05 2.39E−11 KL 197.2 3.69E−036.27E+05 5.89E−09 9.93E−04 1.55E+05 6.39E−10 2.69E−03 2.99E+05 9.00E−09BE 4.99E−04 7.23E+05 6.90E−10 4.80E−08 1.04E+06 4.62E−14 3.02E−033.01E+05 1.01E−08

Example 5 Epitope Mapping

656.35 mAb was epitope mapped and found to bind within KELRCQCIKTYSKP(SEQ ID NO: 54) in human IL-8. Thus in another embodiment, the presentinvention relates to a penta-specific antibody which binds withinepitope of SEQ ID NO:54 of human IL-8.

Example 6 Efficacy Study of a Chimera Antibody Made from 656.35 HavingHeavy Chain Sequence of SEQ ID NO: 56, and Light Chain Sequence of SEQID NO:58. (This Antibody Will be Referred to as the Chimera Antibody)

In Vivo Studies:

An inhaled LPS acute lung inflammatory model was used to examine theability of the a penta-specific antibody to inhibit infiltration ofneutrophils into the lungs of non-human primates (NHPs—cynomolgus).Briefly, NHP (non-human primate) were pre-screened for health andresponsiveness to exogenously added cynomolgus IL-8 (cynoIL-8). SelectedNHP were then subjected to baseline sample collections (blood andbronchoalveolar lavage—BAL) five days prior to the first LPS challenge.LPS challenged consisted of tranquilizing the NHP with a single IMinjection of ketamine HCl (˜10 mg/kg). Once the NHP is sedated,anesthesia is administered, via an intravenous infusion of propofol(˜0.2 mg/kg/min, as necessary). Anesthetized animals are placed on acirculating warm-water heating blanket and/or within a circulatingwarm-air blanket and an ophthalmic lubricant is administered to eacheye. The animals are then intubated and mechanically ventilated forchallenge procedure. A volume regulated Positive Pressure ventilator isused during the procedure (Stoelting Cat/Rabbit ventilatorwww.stoeltingco.com Cat #5019510). Lipopolysaccharide (LPS) challenge isperformed via aerosol inhalation using a DeVilbiss Ultraneb-99ultrasonic nebulizer. Aerosolized LPS is administered for 5 minutes at100 ug/ml. Heart rate, body temperature, and respiration rate aremonitored and recorded during challenge procedures. The initialchallenge confirms that the NHP respond to LPS (increased neutrophilinfiltration into the lungs and elevated cynoIL-8 levels). IndividualNHP responses were confirmed by sample collections at 6 and 24-hourspost challenge.

The initial LPS challenged NHP were then randomly divided into twogroups. Following a 4-week recovery all participating NHP were subjectedto another baseline sample (blood and BAL). Four days post baselinemeasurements the subject groups received IV injection of either avehicle or an injection of 1 mg/kg of the Chimera Antibody. The next daythe NHP were challenged again with inhaled LPS and samples for analysiswere collected 6 and 24-hour post challenge. Following an additionalrecovery period base line samples were collected from the vehicle NHP, 4days later these NHP were treated with a single IV bolus injection ofthe Chimera Antibody at 10 mg/kg. The next day the animals were exposedto an LPS challenge and samples were collected for analysis at 6 and 24hours.

The LPS inhalation is an acute inflammatory model which stimulates anelevation of chemotactic chemokines such at IL-8, which lead toincreased infiltration of neutrophils into the lungs. The primaryefficacy parameter for the Chimera Antibody treatment is inhibition ofinfiltrating neutrophils into the lungs of NHP challenged with LPS.Neutrophil infiltration in this acute inflammatory model occurs duringthe first 24 hours following LPS challenge at which point otherinflammatory processes are becoming more prominent.

Pretreatment of with the Chimera Antibody significantly and dosedependently inhibited neutrophil infiltration into lungs of LPSchallenged NHP. Treatment with the Chimera Antibody also preventedelevation of circulating neutrophils, while not affecting the actualneutrophil function, (i.e. the cells ability to phagocytos). Treatmentalso did not greatly affect other cell types such asmacrophages/monocytes in the lung or circulation. See FIG. 2.

Example 7 Further Studies on Humanized mAbs

Multiple humanized penta-specific antibody constructs have beenproduced, four of which have been shown to bind tightly to, and inhibitall target chemokines. (For each sequence, see Sequence Informationbelow). This analysis consisted of affinity measurements (BiaCore),calcium mobilization assays (in vitro functional assay, FLIPR), andCD11b human and NHP neutrophil stimulation assay (ex vivo functionalassay, flow cytometry).

BiaCore

A Protein A capture method was used to generate kinetics for thehumanised and chimeric constructs as follows:

Protein A is immobilised on a CM5 chip by primary amine coupling inaccordance with the manufactures instructions. Purified humanised orchimeric antibody is captured on the Protein A surface. After capturedefined concentrations of each analyte (IL8, Gro-β and ENA-78) arepassed over the antibody captured surface, a separate capture event isused for each analyte injection. After each injection of analyte thesurface is regenerated by injection of a mild acidic solution, whichremoves the captured antibody but does not significantly affect thecapability of the Protein A to perform another capture event. Aninjection of buffer is also injected over the antibody captured surfaceand this is used for double referencing. The data is analysed using theanalysis software inherent to the machine, using the 1:1 model ofbinding.

Construct ka (1/Ms) kd(1/s) KD(M) Measurements with Gro-beta HcLc7.39E+06 8.16E−04 1.11E−10 HcLc 2.11E+07 0.0011  5.21E−11 HcLc 1.59E+078.88E−04 5.57E−11 HcLc 1.19E+07 6.16E−04 5.17E−11 HcLc 1.43E+07 5.43E−043.79E−11 HcLc 1.33E+07 6.76E−04 5.09E−11 HcLc 1.51E+07 6.73E−04 4.45E−11HcLc 1.49E+07 6.44E−04 4.33E−11 HcLc 2.54E+07 0.001163 4.57E−11 HcLc1.81E+07 0.001058 5.85E−11 HcLc 2.04E+07 8.54E−04 4.18E−11 HcLc 1.82E+075.06E−04 2.78E−11 H0L7 6.99E+06 9.66E−04 1.38E−10 H0L7 1.76E+07 0.0013057.40E−11 H0L7 1.63E+07 0.001039 6.39E−11 H0L7 1.14E+07 0.005616 4.93E−10H0L7 1.20E+07 0.004398 3.66E−10 H0L7 1.42E+07 9.57E−04 6.73E−11 H0L71.76E+07 0.00118 6.72E−11 H0L7 1.63E+07 0.001389 8.52E−11 H0L7 1.12E+070.008212 7.32E−10 H0L7 2.63E+07 0.001546 5.88E−11 H0L7 2.10E+07 9.28E−044.41E−11 H0L8 1.28E+07 0.005462 4.27E−10 H0L8 1.99E+07 0.01065 5.36E−10H0L8 1.19E+07 0.007727 6.52E−10 H0L8 1.18E+07 0.007799 6.60E−10 H0L81.61E+07 0.006903 4.29E−10 H0L8 1.85E+07 0.004586 2.48E−10 H0L101.23E+07 0.001395 1.13E−10 H0L10 1.45E+07 0.001261 8.68E−11 H0L101.44E+07 0.001477 1.03E−10 H0L10 1.28E+07 0.001394 1.09E−10 H0L101.98E+07 0.00144 7.28E−11 H0L10 1.94E+07 0.00109 5.62E−11 H0M0 1.20E+070.001394 1.17E−10 H0M0 1.13E+07 0.007071 6.24E−10 H0M0 1.58E+07 0.0017081.08E−10 H0M0 1.43E+07 0.001829 1.28E−10 H0M0 1.03E+07 0.009588 9.34E−10H0M0 1.85E+07 0.001728 9.35E−11 H0M0 1.81E+07 0.001252 6.91E−11Measurements with IL8 HcLc 3.77E+06 2.34E−04 6.22E−11 HcLc 1.03E+072.87E−04 2.78E−11 HcLc 8.20E+06 2.76E−04 3.37E−11 HcLc 1.04E+07 2.68E−042.58E−11 HcLc 9.44E+06 2.53E−04 2.68E−11 HcLc 9.97E+06 2.30E−04 2.31E−11H0L7 2.74E+06 2.84E−04 1.04E−10 H0L7 8.99E+06 4.00E−04 4.45E−11 H0L77.64E+06 2.84E−04 3.71E−11 H0L7 5.98E+06 0.001167 1.95E−10 H0L7 8.74E+063.52E−04 4.03E−11 H0L7 7.51E+06 3.63E−04 4.83E−11 H0L7 9.10E+06 3.89E−044.28E−11 H0L8 5.90E+06 0.00111 1.88E−10 H0L8 7.89E+06 0.001126 1.43E−10H0L8 6.80E+06 0.001193 1.75E−10 H0L8 7.30E+06 0.001112 1.52E−10 H0L106.86E+06 4.57E−04 6.66E−11 H0L10 7.20E+06 3.67E−04 5.10E−11 H0L107.21E+06 3.96E−04 5.48E−11 H0L10 7.43E+06 3.73E−04 5.02E−11 H0M07.02E+06 3.98E−04 5.67E−11 H0M0 6.40E+06 0.001467 2.29E−10 H0M0 7.96E+064.09E−04 5.14E−11 H0M0 7.97E+06 4.60E−04 5.77E−11 H0M0 7.50E+06 4.18E−045.57E−11 Measurements with ENA78 HcLc 3.67E+06 1.70E−04 4.63E−11 HcLc5.81E+06 1.71E−04 2.94E−11 HcLc 5.11E+06 1.84E−04 3.60E−11 HcLc 5.28E+061.54E−04 2.91E−11 HcLc 4.87E+06 1.56E−04 3.20E−11 HcLc 4.78E+06 1.23E−042.58E−11 H0L7 2.95E+06 2.04E−04 6.93E−11 H0L7 5.28E+06 2.97E−04 5.63E−11H0L7 4.59E+06 2.17E−04 4.74E−11 H0L7 3.03E+06 3.33E−04 1.10E−10 H0L75.11E+06 2.58E−04 5.04E−11 H0L7 4.76E+06 3.07E−04 6.45E−11 H0L7 4.91E+062.77E−04 5.63E−11 H0L8 2.88E+06 2.88E−04 1.00E−10 H0L8 2.82E+06 2.66E−049.41E−11 H0L8 2.95E+06 2.69E−04 9.12E−11 H0L8 3.29E+06 2.48E−04 7.55E−11H0L10 4.07E+06 3.66E−04 8.99E−11 H0L10 4.72E+06 3.40E−04 7.22E−11 H0L104.20E+06 3.12E−04 7.42E−11 H0L10 4.38E+06 2.68E−04 6.13E−11 H0M03.91E+06 3.09E−04 7.89E−11 H0M0 2.97E+06 3.49E−04 1.17E−10 H0M0 4.39E+063.16E−04 7.20E−11 H0M0 4.18E+06 3.34E−04 7.99E−11 H0M0 4.52E+06 2.94E−046.51E−11 Affinity (KD) is determined by examining the association rate(ka) and disassociation rate (kd) of proteins. HcLc refers to chimeraantibody made from 656.35 having heavy chain sequence of SEQ ID NO: 56,and light chain sequence of SEQ ID NO: 58.

Functional Assay: Calcium Mobilization (FLIPR) Using CHO-K1 CXCR2 (w/Gα16)

A microtiter plate based calcium mobilization assay, FLIPR (FluorometricImaging Plate Reader, Molecular Devices, Sunnyvale Calif., [Schroeder,1996]), was used for the functional characterization of the neutralizingeffect of antibodies on ELR+ chemokine induced [Ca²⁺]i-mobilization inCHO-K1 cells transfected with and stably expressing hCXCR2 and Gα16.

On the day prior to assay, cells were plated in 96 well, blackwall,clear bottom plates (Packard View) at a concentration of 40000 cells perwell. After 18 to 24 hours, media was aspirated off cells and replacedwith 100 μl load media containing Eagles Minimal Essential Medium (EMEM)with Earl's salts and L-Glutamine, 0.1% BSA (Serologicals Corporation),4 μM Fluo-4-acetoxymethyl ester fluorescent indicator dye (Fluo-4 AM)and 2.5 mM probenecid. Cells were incubated in this dye containing mediafor 1 hour at 37° C. The dye containing media was then aspirated off thecells and replaced with identical media without Fluo-4 AM and with 0.1%Gelatin (BSA removed) and 2.5 mM probenecid. Cells were incubated for 10min. at 37° C. and then washed 3 times with KRH assay buffer [KrebsRinger Henseleit (120 mM NaCl, 4.6 mM KCl, 1.03 mM KH₂PO₄, 25 mM NaHCO₃,1.0 mM CaCl₂, 1.1 mM MgCl₂, 11 mM Glucose, 20 mM HEPES (pH 7.4)) with0.1% gelatin and 2.5 mM probenecid]. After the final buffer wash, 100 μlKRH assay buffer with 0.1% gelatin and 2.5 mM probenecid was added tocells and warmed to 37° C. for 10 min. before being placed in FLIPRwhere dye loaded cells were exposed to excitation light (488 nm) from a6 watt Argon Laser. [Ca²⁺]i-mobilization was monitored as a result of anincrease in 516 nm emission intensity of Fluo-4 when bound to Ca²⁺.Change in emission intensity is directly related to cytosolic calciumlevels, [Ca²⁺]i. After monitoring baseline for 10 sec., 50 μl of 3×ELR+chemokine, which had been pre-incubated with a concentration range ofantibody, was added to the cell plate and data collected every sec. for1 min., followed by an additional half min. of recording in 3 sec.intervals. Maximal cellular response above basal reading was exportedfor plotting in GraphPad Prism (v4.03).

The IC₅₀ was defined as the concentration of antibody required, duringpre-treatment of 3×EC₈₀ chemokine, to neutralize the CXCR2 mediatedstimulatory effect of an EC₈₀ concentration of the ELR+ chemokine by50%. A secondary cellular response to 25 μM ATP was monitored to testcell viability [Sarau, 1999].

TABLE Inhibition of human ELR+ chemokine induced calcium flux(geometrically meaned IC50, ug/ml) by four humanized mAb constructs. (n= 3) Pre-treated 3X H0L7 H0L8 H0L10 H0M0 EC₈₀ ELR+ IC₅₀ IC₅₀ IC₅₀ IC₅₀Chemokine (ug/ml) 95% C.I. (ug/ml) 95% C.I. (ug/ml) 95% C.I. (ug/ml) 95%C.I. 3 nM hIL8 0.30 0.12-0.72 0.83 0.70-0.98 0.19 0.15-0.22 0.190.15-0.23 10 nM hGROa 1.48 0.98-2.23 3.52 2.74-4.53 1.09 0.58-2.06 1.110.67-1.83 6 nM hGROb 1.04 0.19-5.58 7.93  4.20-14.97 0.66 0.24-1.86 0.990.35-2.76 30 nM hENA-78 2.48 0.78-7.91 2.42 1.27-4.63 1.88 0.83-4.261.75 1.33-2.29 100 nM hGCP-2 11.43  8.19-15.96 40.38 38.45-42.41 11.54 6.30-21.17 13.03  7.20-23.56Schroeder K S, Neagle, B D. FLIPR: a new instrument for accurate, highthroughput optical screening. J. Biomol. Screen. 1996:1-75.Sarau H M, Ames R S, Chambers J, Ellis C, Elshourbagy N, Foley J J etal. Identification, molecular cloning, expression, and characterizationof a cysteinyl leukotrien receptor. Mol Pharmacol. 1999; 56:657-663.

Ex Vivo CD11b Human Neutrophil Stimulation Assay

Flow cytometry was used to examine the humanized penta-specificantibodies' ability to prevent chemokine induced human purifiedneutrophil activation, by following physical cellular changes (size andshape—granulation) and by examining surface activation markers(increased CD11b surface expression). The neutrophil control stimulantFMLP was used to confirm the purified human neutrophils ability toactivate. See FIG. 4.

Sequence Information

Total RNA was extracted from 656.35, 197.2 and 81.1 hybridoma cells,heavy and light variable domain cDNA sequence was then generated byreverse transcription and polymerase chain reaction (RT-PCR). Theforward primer for RT-PCR was a mixture of degenerate primers specificfor murine immunoglobulin gene leader-sequences and the reverse primerwas specific for the antibody constant regions, in this case isotypeIgG2a/κ. Primers were designed according to the strategy described byJones and Bendig (Bio/Technology 9:88, 1991). RT-PCR was carried out induplicate for both V-region sequences to enable subsequent verificationof the correct V-region sequences. The V-region products generated bythe RT-PCR were cloned (Invitrogen TA Cloning Kit) and sequence dataobtained. This process was not successful for 81.1 light variabledomain. Thus, the amino acid sequence shown below (Seq ID NO: 12) wastherefore generated by protein sequencing the light chain isolated on aSDS-polyacrylamide gel run under reducing conditions.Complementarity Determining Regions (CDRs) are underlined.Polynucleotide sequences for heavy and light variable regions (SEQ IDNO: 1 and 3, respectively) for 656.35

SEQ ID NO: 1 CAGGTCCAGTTGCAGCAGTCTGGAGCTGAACTGGTAAGGCCTGGGACTTCAGTGACGATATCCTGTAAGGCTTCTGGCTACACCTTCACTAACTACTGGATAGTTTGGGTCAAACAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATCTTTACTCTGGAGGTGGTTATACTTTCTACAGTGAAAATTTCAAGGGGAAGGCCACACTGACTGCAGACACATCCTCCAGCACTGCCTACATGCACCTCATTAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGATCGGGTTACGACAGAACCTGGTTTGCTCACTGGGGCCAAGGGTCTCTGGTCACTGT CTCTGCA SEQ ID NO: 3GACATCAAGATGACCCAGTCTCCATCCTCCATGTCTGCATCGCTGGGAGAGAGAGTCACTATCACTTGTCAGGCGAGTCAGGACATTGAAAGCTATTTAAGCTGGTATCAGCAGAAACCATGGAAATCTCCTAAGACCCTGATCTATTACGCTACAAGGTTGGCAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGTCAAGATTATTCTCTAACCATCAGCAGCCTGGAGTCTGACGATACAGCAACTTATTACTGTCTACAACATGGTGAGAGCCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGPolypeptide sequences for heavy and light variable regions (SEQ ID NO: 2and 4, respectively) for 656.35

SEQ ID NO: 2 QVQLQQSGAELVRPGTSVTISCKASGYTFTNYWIVWVKQRPGHGLEWIGDLYSGGGYTFYSENFKGKATLTADTSSSTAYMHLISLTSEDSAVYFCARSG YDRTWFAHWGQGSLVTVSASEQ ID NO: 4 DIKMTQSPSSMSASLGERVTITCQASQDIESYLSWYQQKPWKSPKTLIYYATRLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPPTFGA GTKLELKRPolypeptide sequences for heavy chain CDRs (SEQ ID NO: 13, 14, 15,respectively) for 656.35

NYWIV SEQ ID NO: 13 DLYSGGGYTFYSENFKG SEQ ID NO: 14 SGYDRTWFAH SEQ IDNO: 15Polypeptide sequences for light chain CDRs (SEQ ID NO: 16, 17, 18) for656.35

QASQDIESYLS SEQ ID NO: 16 YATRLAD SEQ ID NO: 17 LQHGESPPT SEQ ID NO: 18Polynucleotide sequences for heavy chain CDRs (SEQ ID NO: 31, 32, 33)for 656.35

SEQ ID NO: 31 AACTACTGGATAGTT SEQ ID NO: 32GATCTTTACTCTGGAGGTGGTTATACTTTCTACAGTGAAAATTTCAAGG GG SEQ ID NO: 33TCGGGTTACGACAGAACCTGGTTTGCTCACPolynucleotide sequences for light chain CDRs (SEQ ID NO:34, 35, 36) for656.35

CAGGCGAGTCAGGACATTGAAAGCTATTTAAGC SEQ ID NO: 34 TACGCTACAAGGTTGGCAGATSEQ ID NO: 35 CTACAACATGGTGAGAGCCCTCCCACG SEQ ID NO: 36Polynucleotide sequences for heavy and light variable regions (SEQ IDNO: 5 and 7, respectively) for 197.2

SEQ ID NO: 5 GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACATGAACTGGGTGAAGCAGAGCAATGGAAAGAGCCTTGAGTGGATTGGAGTAATTAATCCTAAGTATGGTACTACTAGTTACAATCAGAAGTTCAAGGGCAAGGCCACGTTGACTGTAGACCAATCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATCACTGTGCAAGAGGAATGGGACTCCTCTTTGGTATGGACTACTGGGGCCAAGGAACCTCTGTCACCGT CTCCTCA SEQ ID NO: 7GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCARAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACGGGGCATCCACTAGGAAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTTTCCGTGCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGPolypeptide sequences for heavy and light variable regions (SEQ ID NO: 6and 8, respectively) for 197.2

SEQ ID NO: 6 EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGVINPKYGTTSYNQKFKGKATLTVDQSSNTAYMQLSSLTSEDSAVYHCARGM GLLFGMDYWGQGTSVTVSSSEQ ID NO: 8 DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRKSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSF PCTFGGGTKLEIKRPolypeptide sequences for heavy chain CDRs (SEQ ID NO: 19, 20, 21,respectively) for 197.2

DYNMN SEQ ID NO: 19 VINPKYGTTSYNQKFKG SEQ ID NO: 20 GMGLLFGMDY SEQ IDNO: 21Polypeptide sequences for light chain CDRs (SEQ ID NO: 22, 23, 24) for197.2

KSSQSLLNSGNQKNYLA SEQ ID NO: 22 GASTRKS SEQ ID NO: 23 QNDHSFPCT SEQ IDNO: 24Polynucleotide sequences for heavy chain CDRs (SEQ ID NO: 37, 38, 39)for 197.2

SEQ ID NO: 37 GACTACAACATGAAC SEQ ID NO: 38GTAATTAATCCTAAGTATGGTACTACTAGTTACAATCAGAAGTTCAAG GGC SEQ ID NO: 39GGAATGGGACTCCTCTTTGGTATGGACTACPolynucleotide sequences for light chain CDRs (SEQ ID NO:40, 41, 42) for197.2

SEQ ID NO: 40 AAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTG GCC SEQID NO: 41 GGGGCATCCACTAGGAAATCT SEQ ID NO: 42CAGAATGATCATAGTTTTCCGTGCACGPolynucleotide sequence for heavy variable region (SEQ ID NO: 9) for81.1

SEQ ID NO: 9 GAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGAGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCTTTCACTGTCTACGGCATGAACTGGGTGAGACAGAGCAATGGAAAGAGCCTTGAATGGATTGGAAATTTTGATCCTTACTTTAGTGTCACTTCCTACAACCAGAAGTTCCAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAGAACCTCACATCTGAAGACTCTGCAGTCTATTTCTGTGCAAGAGGGAGCTGGGAAACCATTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTC TGCAPolypeptide sequence for heavy variable region (SEQ ID NO: 10) for 81.1

SEQ ID NO: 10 EVQLQQSGPELEKPGASVKISCKASGYSFTVYGMNWVRQSNGKSLEWIGNFDPYFSVTSYNQKFQDKATLTVDKSSSTAYMQLKNLTSEDSAVYFCARGS WETIFAYWGQGTLVTVSAPolypeptide sequences for heavy chain CDRs (SEQ ID NO: 25, 26, 27,respectively) for 81.1

VYGMN SEQ ID NO: 25 NFDPYFSVTSYNQKFQD SEQ ID NO: 26 GSWETIFAY SEQ ID NO:27Polynucleotide sequences for heavy chain CDRs (SEQ ID NO: 43, 44, 45)for 81.1

SEQ ID NO: 43 GTCTACGGCATGAAC SEQ ID NO: 44AATTTTGATCCTTACTTTAGTGTCACTTCCTACAACCAGAAGTTCCAGG AC SEQ ID NO: 45GGGAGCTGGGAAACCATTTTTGCTTACPolypeptide sequence for light variable region (SEQ ID NO: 12) for 81.1

QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHVFG GGTKLTVLQPKPolypeptide sequences for light chain CDRs (SEQ ID NO: 28, 29, and 30,respectively) for 81.1

RSSTGAVTTSNYAN SEQ ID NO:28 GTNNRAP SEQ ID NO:29 ALWYSNHV SEQ ID NO:30A chimera* polynucleotide sequence (variable heavy region+codonoptimised IgG1)

SEQ ID NO: 55 CAGGTCCAGTTGCAGCAGTCTGGAGCTGAACTGGTAAGGCCTGGGACTTCAGTGACGATATCCTGTAAGGCTTCTGGCTACACCTTCACTAACTACTGGATAGTTTGGGTCAAACAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATCTTTACTCTGGAGGTGGTTATACTTTCTACAGTGAAAATTTCAAGGGGAAGGCCACACTGACTGCAGACACATCCTCCAGCACTGCCTACATGCACCTCATTAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGATCGGGTTACGACAGAACCTGGTTTGCTCACTGGGGCCAAGGGTCACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCA AGchimera* polypeptide sequence (variable heavy region+codon optimisedIgG1)

SEQ ID NO: 56 QVQLQQSGAELVRPGTSVTISCKASGYTFTNYWIVWVKQRPGHGLEWIGDLYSGGGYTFYSENFKGKATLTADTSSSTAYMHLISLTSEDSAVYFCARSGYDRTWFAHWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKA chimera* polynucleotide sequence (variable light region+codonoptimised human cK)

SEQ ID NO: 57 GACATCAAGATGACCCAGTCTCCATCCTCCATGTCTGCATCGCTGGGAGAGAGAGTCACTATCACTTGTCAGGCGAGTCAGGACATTGAAAGCTATTTAAGCTGGTATCAGCAGAAACCATGGAAATCTCCTAAGACCCTGATCTATTACGCTACAAGGTTGGCAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGTCAAGATTATTCTCTAACCATCAGCAGCCTGGAGTCTGACGATACAGCAACTTATTACTGTCTACAACATGGTGAGAGCCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCChimera* polypeptide sequence (variable light region+codon optimisedhuman cK)

SEQ ID NO: 58 DIKMTQSPSSMSASLGERVTITCQASQDIESYLSWYQQKPWKSPKTLIYYATRLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECChimera* refers to chimeric antibody made from murine 656.35 antibody

H0 DNA sequence of mature heavy chain SEQ ID NO: 11CAGGTGCAGCTCGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGGGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATCGTGTGGGTCAGGCAGGCCCCCGGCCAGGGACTGGAGTGGATGGGCGACCTGTATAGCGGCGGCGGCTACACCTTCTACAGCGAGAACTTCAAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGAGCGGCTACGACAGGACTTGGTTTGCTCACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG H0 protein sequence ofmature heavy chain SEQ ID NO: 46QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIVWVRQAPGQGLEWMGDLYSGGGYTFYSENFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGYDRTWFAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK L7 DNA sequence ofmature light chain SEQ ID NO: 47GATATCCAGATGACCCAGAGCCCTAGCTCCCTCAGCGCATCAGTCGGCGACAGAGTGACAATCACCTGCCAGGCATCCCAGGACATCGAGTCTTACCTGAGCTGGTACCAGCAGAAGCCCGGAAAGGCCCCAAAGCTCCTGATCTACTACGCCACTCGGCTGGCAgacGGCGTGCCTAGCAGGTTCTCCGGCTCAGGGTCTGGGACAGACTTCACCCTGACCATCAGCTCACTGCAGCCCGAGGATTTCGCCACCTACTACTGTCTGCAGCACGGAGAGAGCCCCCCAACCTTTGGCCAGGGAACCAAGCTGGAGATCaagCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC L7 protein sequence of maturelight chain SEQ ID NO: 48DIQMTQSPSSLSASVGDRVTITCQASQDIESYLSWYQQKPGKAPKLLIYYATRLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHGESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC L8 DNAsequence of mature light chain SEQ ID NO: 59GATATCCAGATGACCCAGAGCCCTAGCTCCCTCAGCGCATCAGTCGGCGACAGAGTGACAATCACCTGCCAGGCATCCCAGGACATCGAGTCTTACCTGAGCTGGTACCAGCAGAAGCCCGGAAAGGCCCCAAAGCTCCTGATCTACTACGCCACTCGGCTGGCAgacGGCGTGCCTAGCAGGTTCTCCGGCTCAGGGTCTGGGACAGACTTCACCttcACCATCAGCTCACTGCAGCCCGAGGATatcGCCACCTACTACTGTCTGCAGCACGGAGAGAGCCCCCCAACCTTTGGCCAGGGAACCAAGCTGGAGATCaagCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC L8 protein sequence of maturelight chain SEQ ID NO: 60DIQMTQSPSSLSASVGDRVTITCQASQDIESYLSWYQQKPGKAPKLLIYYATRLADGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHGESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC L10DNA sequence of mature light chain SEQ ID NO: 61GATATCCAGATGACCCAGAGCCCTAGCTCCCTCAGCGCATCAGTCGGCGACAGAGTGACAATCACCTGCCAGGCATCCCAGGACATCGAGTCTTACCTGAGCTGGTACCAGCAGAAGCCCGGAAAGGCCCCAAAGCTCCTGATCTACTACGCCACTCGGCTGGCAgacGGCGTGCCTAGCAGGTTCTCCGGCTCAGGGTCTGGGcagGACtacACCCTGACCATCAGCTCACTGCAGCCCGAGGATTTCGCCACCTACTACTGTCTGCAGCACGGAGAGAGCCCCCCAACCTTTGGCCAGGGAACCAAGCTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC L10 protein sequence ofmature light chain SEQ ID NO: 62DIQMTQSPSSLSASVGDRVTITCQASQDIESYLSWYQQKPGKAPKLLIYYATRLADGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQHGESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC M0 DNAsequence of mature light chain SEQ ID NO:63GAGATCGTGCTGACCCAGTCTCCCGCCACCCTGTCACTGTCTCCCGGCGAAAGGGCAACCCTGAGCTGCCAGGCCAGCCAGGACATCGAGAGCTACCTGAGCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACTACGCCACCAGGCTGGCCGACGGCATTCCCGCCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACTCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCTGCAGCACGGCGAGAGCCCTCCCACCTTCGGCCAGGGCACCAAGCTCGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC M0 protein sequence of maturelight chain SEQ ID NO: 64EIVLTQSPATLSLSPGERATLSCQASQDIESYLSWYQQKPGQAPRLLIYYATRLADGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQHGESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

1. An antibody comprising heavy and light chains comprising the aminoacid sequences of SEQ ID NO:46 and SEQ ID NO:48, respectively.
 2. Anantibody comprising heavy and light chains comprising the amino acidsequences of SEQ ID NO:46 and SEQ ID NO: 62, respectively.
 3. Anantibody comprising heavy and light chains comprising the amino acidsequences of SEQ ID NO:46 and SEQ ID NO: 60, respectively.
 4. Anantibody comprising heavy and light chains comprising the amino acidsequences of SEQ ID NO:46 and SEQ ID NO: 64, respectively